Immunomodulatory Mechanisms of Endoplasmic Reticulum Stress in the Tumor Immune Microenvironment and Prediction of Treatment Response in HER2-Positive Breast Cancer

Immunomodulatory Mechanisms of Endoplasmic Reticulum Stress in the Tumor Immune Microenvironment and Prediction of Treatment Response in HER2-Positive Breast Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunomodulatory Mechanisms of Endoplasmic Reticulum Stress in the Tumor Immune Microenvironment and Prediction of Treatment Response in HER2-Positive Breast Cancer Barbora Vavrušáková, Lenka Krejčí, Lukáš Pečinka, Iveta Selingerová, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8709304/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract HER2-positive breast cancer (BC) is an aggressive subtype with poor long-term outcomes in advanced disease, largely due to resistance to HER2-targeted therapies. Within the tumor-immune microenvironment (TIME), tumor and immune cells face diverse stressors, including endoplasmic reticulum stress (ERS). ERS activates the Unfolded Protein Response (UPR), which aims to restore homeostasis or trigger apoptosis. In BC cells, chronic UPR activation promotes malignant progression, while in immune cells ERS impairs function and weakens antitumor immunity. Given the relatively high immune infiltration in HER2-positive BC, preserving immune competence is crucial for therapeutic efficacy. This study aims to elucidate TIME-associated molecular mechanisms in HER2-positive BC using in-vitro model and patient tumor samples analysis from 21 patients and to develop predictive models of treatment response using liquid biopsy-based mass spectrometry (MS) approach (21 patients, 15 healthy donors). ERS modulation was shown to significantly affect cancer–immune cell interactions in co-culture models. Key ERS-related genes were characterized at the transcriptional, translational, and spatial levels. These findings were used to model the effects of immunomodulatory therapies in vitro . Analysis of patient samples revealed distinct TIME patterns associated with treatment response. Serum-based MALDI-TOF MS enabled the development of a predictive model that discriminated responders from non-responders. Modulating ERS within the TIME represents a promising strategy to enhance immune-mediated antitumor activity in HER2-positive BC. This study provides a detailed molecular characterization of the HER2-positive BC TIME and presents an MS- and machine learning-based predictive model for patient stratification. HER2-positive Breast Cancer Endoplasmic Reticulum Stress Unfolded Protein Response Tumor Immune Microenvironment MALDI TOF Mass Spectrometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction HER2-positive breast cancer (BC) accounts for 15–20% of all cases. The introduction of targeted therapies such as trastuzumab and pertuzumab has significantly improved patient outcomes [ 1 ]. HER2-activated BCCs are more vulnerable to damage to the inhibition of HER2 signaling, particularly in context of radiotherapy. HER2 gene amplification/protein overexpression predicts poor prognosis and response to certain types of chemotherapy [ 2 , 3 ] and correlates with a significantly shorter disease-free survival. [ 4 ] The tumor immune microenvironment (TIME) contains non-transformed host cellular components of the tumor mass residing within the tumor region [ 5 ]. The BC TIME plays an important role in tumor development, progression and metastasis, anti-tumor immunity [ 6 ], is highly plastic, and undergoes constant changes and stage-specific adaptations depending on various cancer cell-intrinsic and extrinsic factors [ 7 , 8 ]. Throughout the progression of BC, the stroma undergoes significant changes, including infiltration of immune cells, inflammation, angiogenesis, and remodeling of the ECM [ 9 – 11 ]. Within the local TIME, immune cells can acquire functional plasticity and adapt, which allows them to play dual role as either pro or anti-tumor agent [ 10 ]. Cancer cells employ various immune evasion strategies that can be enhanced by TIME and thus can suppress the infiltration, activation, and effector functions of immune cells [ 10 , 11 ]. Since TIME elements are involved in various stages of tumorigenesis, the TIME appears to be an attractive therapeutic target. Tumor-infiltrating lymphocytes (TILs) in TIME regulate the anti-tumor immunity, immunosuppression, immunotherapy effectiveness, invasiveness and metastasis, and chemo- and radiotherapy resistance [ 12 ]. A variety of stress factors can impair endoplasmic reticulum (ER) homeostasis leading to the state of the endoplasmic reticulum stress (ERS) [ 13 ]. Homeostasis disruption leads to the accumulation of abnormal proteins in the ER lumen, which sequester the ER-chaperone BiP. This triggers specific signaling pathways, collectively known as the unfolded protein response (UPR), which significantly influence cell fate. UPR consists of three main UPR signaling pathways , controlled by the proteins IRE1α, PERK, and ATF6 (Fig. 1 ). These pathways help restore normal functions following cell injuries. IRE1α allows to activate the XBP1 gene, which helps to restore protein balance by promoting the production of chaperone proteins and proteins involved in breaking down misfolded proteins [ 14 , 15 ]. PERK halts global protein synthesis by phosphorylating eIF2α. This reduces protein production but allows specific proteins like ATF4 to be made. ATF4 then triggers the production of proteins related to oxidative stress and autophagy [ 16 ]. Lastly, ATF6 promotes the transcription of genes involved in chaperone production, lipid metabolism, and UPR regulation, helping reduce ERS. In response to ERS, the UPR works to protect the cell by producing chaperone proteins, regulating lipid metabolism, and activating autophagy and antioxidant responses. However, depending on the extent of cellular damage, the UPR can activate either pro-survival or pro-apoptotic mechanisms. In case of persisting or severe ERS, the UPR can trigger cell death. This occurs through pathways like ATF4 and ATF6 activating the pro-apoptotic CHOP protein, IRE1α activating apoptosis via TRAF2 and JNK, or IRE1α activating caspases. There is abundant evidence that ERS-mediated response pathway is strictly associated with the pathogenesis of many human diseases including cancer [ 17 ]. In healthy cells, UPR pathways aim to resolve cell injury or initiate cell death, depending on the severity of the damage and cellular/tissue status. However, cancer cells often manipulate these stress mechanisms to support their survival by reprogramming their metabolism, activating oxidative stress responses, promoting autophagy, and inhibiting apoptosis and senescence. Functional studies have demonstrated that the tumoral UPR actively communicates with stromal cells, influencing cancer progression in a cell-nonautonomous manner. Transmissible ERS (TERS) was initially described as a form of UPR-mediated intercellular communication between cancer cells and normal cell types [ 18 ]. Studies have shown that ER-stressed cancer cells are capable of activating the UPR in macrophages and other immune cells via the secretion of soluble factors into the culture media. This can lead to suppression of antitumor immune responses [ 19 – 21 ]. Cancer cells were shown to activate the UPR of surrounding cancer cells, enhancing resistance to standard anticancer drugs [ 22 ]. UPR in immune cells plays a crucial immunosuppressive role within the TIME, supporting cancer progression, immune evasion, and therapy resistance [ 21 , 23 ]. Tumor cells manipulate UPR pathways—particularly PERK and IRE1α-XBP1 signaling—to reprogram immune cells, leading to impaired antigen presentation, dysfunctional T cell responses, and diminished dendritic cell (DCs) activity [ 24 – 27 ]. This reprogramming also fosters immunosuppressive phenotypes in tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) [ 19 , 20 , 28 , 29 ]. Targeting UPR-mediated immunomodulatory mechanisms offers promising avenues for novel cancer treatment strategies. Of note, the UPR has been targeted in noncancer cells (i. e., immune cells or whole body), observing important anticancer effects. These findings raise questions about the biological significance of TERS in cancer, emphasizing the need for further investigation to confirm the existence and impact of cell-to-cell UPR transmission under physiological conditions. Blocking ERS sensors with small molecules can effectively suppress tumor growth by inhibiting angiogenesis, which was observed in various cancer types including BC [ 30 , 31 ]. Employing selective UPR inhibitors has the potential to enhance the function of TAMs and DCs, thereby mitigating the immunosuppressive environment within tumors. This strategy could be especially advantageous when combined with immune checkpoint inhibitors, potentially boosting their effectiveness. Supporting this idea, pharmacological inhibition of IRE1 and PERK signaling pathways in TAMs improved anti-PD-1 therapy efficacy by reducing the immunosuppressive phenotype of macrophages, lowering PD-L1 expression, promoting T cell activity and delayed tumor growth [ 21 , 26 , 28 ]. Targeting BiP reduces CD47 expression, leading to increased tumor macrophage infiltration and reduced resistance to anti-estrogen therapy. Co-expression of BiP and CD47 correlates with poor prognosis in BC patients, highlighting crosstalk between the UPR and immune regulation in shaping therapeutic outcomes [ 32 ]. This study explores novel strategies to enhance the antitumor activity of immune cells by modulating ERS within the TIME, aiming to overcome immunosuppressive mechanisms and ultimately improve the effectiveness of anticancer therapies in HER2-positive BC. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor buffy coats by density gradient centrifugation and cultured in complete OpTmizer medium supplemented with FBS, L-glutamine, antibiotics, and IL-2. The HER2-positive breast cancer cell line SK-BR-3 was obtained from ATCC, authenticated by STR profiling, and maintained in high-glucose DMEM with FBS and antibiotics. For co-culture experiments, SK-BR-3 cells were seeded and allowed to adhere for 24 h, after which PHA-activated PBMCs were added at a 2:1 ratio. Co-cultures were treated with ERS modulators (TUDCA, tunicamycin), anti-HER2 trastuzumab, anti-PD-L1 atezolizumab, or their combinations at defined working concentrations. Cell viability was assessed by MTT assay, measuring formazan absorbance at 595 nm. Cell migration was evaluated using a scratch wound assay with live-cell imaging over 24 h. Gene expression was analyzed by RT-qPCR following phenol-chloroform RNA isolation, cDNA synthesis, and amplification using ACTB as a reference gene. Protein expression was examined by SDS-PAGE and Western blotting, with β-actin as a loading control. Immunofluorescence staining of in vitro co-cultures was performed to visualize target proteins, actin cytoskeleton, and nuclei using fluorescence microscopy. Retrospective clinical analyses included HER2-positive breast cancer patients treated with anti-HER2 therapy plus chemotherapy, with ethical approval and informed consent. Patients were stratified by early failure, and survival outcomes were analyzed using Kaplan–Meier estimates and log-rank tests. Tumor FFPE sections were processed for immunofluorescence microscopy. Serum proteomic profiling was conducted by MALDI-TOF MS following protein extraction, with spectra analyzed using multivariate statistical methods to identify discriminative features between patient groups and healthy controls. For extended version of Methods see Supplementary Information. Results Impact of ERS modulation on BCCs viability and motility in vitro The effects of ERS modulation within TIME on cancer and immune cells were evaluated in both mono- and co-cultures of PBMCs and BCCs treated with TUDCA, a chemical chaperone attenuating general ERS, and Tun, as an established inducer of ERS inhibiting the N-glycosylation machinery. Effective concentrations were established to ensure that TUDCA exerted its positive ERS modulating effect primarily on immune cells rather than tumor cells. Tun led to a distinct decrease in cell viability both in mono- and co-culture conditions. (Fig. 2 A). BC in co-culture exhibited slightly higher motility in control conditions, whereas under Tun, migration was slightly suppressed (Fig. 2 B). ERS modulation can drive gene and protein expression changes specifically in immune cells To link the pilot observations with underlying molecular mechanisms, cells were treated for 24 h with selected concentrations of ERS modulators and treatment-associated gene expression alterations in UPR and immune markers were assessed. The effect of Tun in UPR activation was evident in monoculture and co-culture; Tun increased the expression of UPR markers, particularly the major regulator of the UPR chaperone HSPA5/BiP and the proapoptotic transcription factor DDIT3/CHOP. The effect of TUDCA was particularly pronounced in PBMCs, where UPR signaling was attenuated. Differences in expression were also observed between mono- and co-cultures in response to modulating conditions, suggesting that PBMCs were targeted specifically. Given the role of EMT in cancer, the levels of EMT markers were examined. In SK-BR-3, changes in gene expression indicate activation of EMT processes under co-culture conditions. The effect of ERS on immune function was evaluated in co-cultures of BCCs and PBMCs. In PBMCs monoculture, Tun led to a slight increase in CD4 transcription. To cover population variability, PBMCs were isolated from different donors for each biological replicate. Therefore, gene expression of CD3E , CD4 , CD8A , IL2RA , and ENTPD1 varied between biological replicates, depending on the donor of PBMCs (Fig. 2 C-E). To describe these effects on protein level , immunoblotting was performed to visualize ERS associated proteins and immune markers of both PBMCs and BCCs after 48 h treatment with selected concentrations of ERS modulators. SK-BR-3 cells exhibited signs of UPR activation even under control conditions, with increases in BiP, CHOP, and IRE1α protein levels upon Tun treatment and further modulation in co-culture. Tun exposure also led to decreased levels of immune regulatory proteins PD-L1, PD-1, and FOXP3. In PBMCs monoculture, Tun treatment elevated BiP, CHOP, and IRE1α levels, which were subsequently reduced—particularly BiP—by TUDCA. Tun additionally upregulated CD8 protein levels while downregulating FOXP3. Notably, immune marker expression in PBMCs varied depending on the donor (Fig. 2 F). To monitor the spatial localization and abundance of BiP and CHOP in in vitro co-cultures, we employed IF staining (Fig. 3 ). In SK-BR-3 cells, elevated levels of BiP and CHOP proteins were present in the control condition, corresponding to results from immunoblotting. Tun induced only a partial increase in BiP and CHOP protein levels according to image analysis, while addition of TUDCA in co-culture further enhanced this effect (Fig. 3 B). Similarly, BiP was present only in the cytoplasm, whereas CHOP was localized in all conditions in both the cytoplasm and the nucleus, with a shift to the nucleus upon Tun treatment. The effect of ERS modulation was also evident in PBMCs, where we detected increased levels of CHOP protein, most abundantin the presence of Tun, while this effect was successfully reversed by treatment with TUDCA. BiP protein was almost undetectable in PBMCs using this method. ERS modulation impact on the effectiveness of targeted immunomodulatory therapy in vitro Current strategies for HER2-positive BC include treatment with monoclonal antibodies such as Her and Tec. To simulate immunomodulatory conditions, these agents were applied to SK-BR-3 cells in combination with ERS modulators. A significant reduction in tumor cell viability of SK-BR-3 was observed following treatment with Tun and its combinations with Her / Tec, with the most pronounced effects seen in co-culture with PBMCs (Fig. 4 A-B). Combined treatment of Tun with Her or Tec increased gene expression of UPR and apoptotic markers(mainly HSPA5 , DDIT3 , ERN1, XBP1s , and BCL2 ) in PBMCs. However, transcriptional activation of UPR markers was attenuated by the treatment with TUDCA. Importantly, TUDCA did not markedly affect expression of these markers in SK-BR-3 (Fig. 4 C-F). At protein level, increased expression of BiP, CHOP, and XBP1s was detected in PBMCs after the co-treatment of Tun and Her/Tec, with levels reduced upon TUDCA addition (mainly with Tec). Similarly, these UPR markers were upregulated in tumor cells primarily in response to combined treatments. TUDCA moderately decreased these protein levels, particularly CHOP. Notably, HER2 protein levels were reduced following Her treatment, in the presence of PBMCs (Fig. 4 G-H). In terms of immune activation, combined treatments of Tun and immunomodulation resulted in increased transcription of immune-related genes CTLA4 , IL2 , IFNG , CD4 , CD8A , and ENTPD1 in PBMCs. TUDCA co-treatment reduced expression of CTLA4 , IL2 , IFNG , and CD4 (Fig. 4 C-F). Combination of Tun and immunomodulatory treatment decreased protein levels of CD3, CD4, CD8, and FOXP3, with partial restoration observed upon TUDCA addition (Fig. 4 G-H). Similarly to other PBMCs-associated results, immune responses varied among PBMCs donors. Collectively, these findings indicate that modulation of ERS significantly influences tumor and immune cell interaction in vitro . Tun-induced ERS amplifies UPR markers and suppresses immune-related proteins, while TUDCA mitigates several of these effects, suggesting a regulatory role. To apply findings to the clinical setting, analyses were extended to a cohort of patients with HER2-positive BC. A total of 21 patients (median age 59 years, range 21–76 years) were enrolled in this study. Of these, 11 (52%) were diagnosed with stage II and 10 (48%) with stage III disease, and all patients but one had nodal involvement. Eight patients underwent primary surgery, while the remaining 13 received neoadjuvant therapy. Anti-HER2 therapy consisted of trastuzumab alone in 14 (67%) patients and trastuzumab plus pertuzumab in the remainder. Overall, 70% of patients completed at least 48 weeks of anti-HER2 therapy. Early failure occurred in 9 patients, more frequently in stage III disease (67%), although the difference was not statistically significant. No other clinicopathological characteristics were associated with early failure. During a median follow-up of 80.5 months, the 5-year OS rate was 75% (95% CI 58–97%), with 42% in EF patients and 100% in non-EF (p = 0.009). The 3-year and 5-year EFS rates of 57% (95% CI 39–83%) and 47% (95% CI 30–74%), respectively. Non-EF patients showed lower occurrence and relative intensity of UPR markers, whereas immune related markers were measured at higher levels than in the EF group. These findings largely correlated with the probability of EFS in these patients, where CHOP, CD3 and CD4 markers showed the most promising predictive potential (Fig. 5 A-B, Suppl. Figure 1) . Unsupervised PCA based on MALDI-TOF MS data showed a distinct separation between the CTR and EF groups, with partial overlap of non-EF samples ( Suppl. Figure 2A ). The supervised PLS-DA and OPLS-DA (for two clinical groups) performed on all samples confirmed these results, demonstrating clear separation of CTR and EF groups (Fig. 5 C), partial separation of non-EF and EF (Fig. 5 D), and distinct clustering of CTR and non-EF and EF groups (Fig. 5 E). All selected ML prediction models except DT demonstrate the capacity to discriminate between CTR and EF groups with a high accuracy, reaching a maximum of 99%, CI: 98–100% for PLS-DA algorithms (Fig. 5 F). For differentiation between EF and non-EF groups, RF-based models achieved the optimal performance, with a maximum accuracy of 81%, CI: 75–87% (Fig. 5 G). Including CTR samples slightly reduced the accuracy to 80%, CI: 75–85% for the RF-based model (Fig. 5 H). Two signals, detected at m/z 2863.4 and 8926.8 Da, significantly contributed to the RF-based model ( Suppl. Figure 2B-C ). The 8926.8 Da signal enables discrimination between the EF and non-EF groups, whereas the 2863.4 Da signal distinguishes the CTR group from both patient groups. Discussion Tumor cells, through their aberrant growth and metabolism, create a particularly inhospitable environment characterized by various stress factors such as low pH, nutrient deprivation, and hypoxia. These factors contribute to the disruption of ER homeostasis in many cell types within TIME, leading to ERS and the activation of UPR signaling pathways. In most tumor cells, the continuous activation of the UPR facilitates malignant progression by promoting angiogenesis, proliferation, and tolerance to hypoxia. In contrast, excessive ERS in immune cells impairs their function, thereby weakening antitumor immunity [ 23 , 33 ]. HER2-positive BC is characterized by elevated levels of TILs within TIME [ 34 ]. These TILs significantly contribute to the therapeutic efficacy of monoclonal anti-HER2 antibodies [ 35 ]. However, the impairment of immune cell function due to excessive ERS is highly undesirable, as it limits their ability to target tumor cells. Here we determine whether modulating ERS in immune cells within the TIME could enhance their capacity to target tumor cells effectively. The interaction of PBMCs with BCCs induced changes in UPR signaling in vitro , together with an increase in the expression of the anti-apoptotic gene BCL2 , particularly upon Tun treatment, reflecting stress adaptation and survival signaling [ 36 ]. These results suggest resistance of BCCs to immune response and reduced apoptotic signalling preventing PBMCs from mitigating BC in vitro. Additionally, SK-BR-3 cells exhibited an increase in the expression of the pro-apoptotic gene BAX , mainly under Her treatments. Tun induced ERS and activated UPR signaling, profoundly reduced cell viability under all tested conditions and slightly suppressed cellular motility in co-culture conditions in SK-BR-3 cells. In these cells, Tun primarily influenced the DDIT3 gene, which induces apoptosis and is associated with longer patient survival when highly expressed [ 37 , 38 ]. In SK-BR-3 cells, continuous activation of the UPR signaling was observed even under control conditions. Additionally, these cells exhibited higher levels of HER2 protein. This suggests that the SK-BR-3 cells correspond to a more aggressive tumor type, strongly driven by the HER2 growth factor. The persistent activation of UPR signaling pathways may enable these BCCs to better adapt to changing conditions and leverage this adaptability to their advantage. When monitoring the effect of ERS modulators on the immune response to BCCs, the biological variability of individual PBMCs donors was highly evident, especially on the immune profile. TUDCA consistently attenuated UPR signaling in PBMCs, primarily reflected in decreased levels of HSPA5/BiP, DDIT3 /CHOP, ERN1 /IRE1α, and XBP1s /XBP1s. Notably, elevated levels of these proteins in TILs are typically associated with diminished infiltration into the TIME, disease progression, metastasis, and poorer clinical outcomes in patients with ovarian cancer [ 27 , 29 , 39 ]. In SK-BR-3 cells, TUDCA partially suppressed Tun-induced UPR marker expression, with strongest attenuation in co-culture, suggesting that the apparent decrease in total protein levels originates from PBMCs rather than from BCCs. Furthermore, TUDCA led to reduced T-cell activation and regulatory markers in PBMCs, most prominently FOXP3 and CD4 , as well as a partial reduction in CTLA4 , IL2 , IFNG , and ENTPD1 [ 40 , 41 ]. This work also investigated the potential effect of the drug TUDCA in promoting the efficacy of anti-HER2 therapy. TUDCA in combination with the drug Her caused an even more pronounced attenuation of ERS signalling in PBMCs, particularly decreasing HSPA5, DDIT3 , and ERN1 expression, thereby limiting pro-apoptotic UPR activation. TUDCA reduced Tun-induced transcription of IL2 in both mono- and co-culture settings (predominantly produced by CD4 + TILs in response to antigenic stimulation). IL2 promotes TILs proliferation and differentiation, and its administration promotes disease remission in some cancer patients [ 42 ]. In the Tec arm, TUDCA moderately reduced overall UPR marker expression in both BCCs and PBMCs, but did not significantly enhance immune activation. Not only the abundance of individual stress markers but also their localization, suggestive of their characteristic activity, was examined. The localization of the UPR proteins BiP and CHOP was specific. BiP was present in all conditions only in the cytoplasm, specifically as granules in the ER [ 37 ]. However, CHOP protein was translocated from the cytoplasm to the nucleus upon ERS, where it functions as a pro-apoptotic transcription factor, and this translocation is thus indicative of active signaling [ 43 ]. CHOP remained partly nuclear even under control conditions, supporting persistent UPR activation in SK-BR-3 cells. Collectively, these findings indicate that modulation of ERS significantly influences tumor and immune cell responses to targeted immunomodulatory therapy in vitro . Tun promotes UPR activation and suppresses immune-related proteins, while TUDCA mitigates several of these effects, suggesting a regulatory role. Her reduced HER2 protein levels, particularly in co-culture conditions. We analyzed the molecular background of retrospectively obtained HER2-positive BC tumor tissue samples to identify the role of ERS in BC TIME and its’ effect on infiltrated immune cells. We observed a positive correlation between EF and lower TILs infiltration (CD3+, CD4+, and CD8+) in the TIME. The number of TILs in tumor tissue was also correlated with treatment outcome by Rathore et al. , who described that infiltration of CD3+, CD4+, and CD8 + TILs was significantly associated with better prognosis of BC patients and reduced risk of disease relapse [ 44 ]. Similarly, we proved the level of CD4 + TILs to be a good prognostic marker of overall EFS in the performed analysis. In this study, we also correlated the level of UPR markers in TIME with EFS. A higher BiP and CHOP signal in whole tissue, as well as in the vicinity of CD3 + TILs, was observed in EF patients compared to the non-EF group. We can summarize that longer EFS is associated with more TILs in the TIME, their higher activity against BCCs and lower UPR activation in the TIME. Finally, we developed predictive model based on MALDI-TOF MS analysis of peripheral blood serum. We showed that spectral data combined with PCA and supervised PLS/OPLS-DA effectively separated CTR and EF groups, while non-EF group displayed partial overlap with EF group. ML predictive models, particularly RF, k-NN, ANN, and PLS-DA, achieved near-perfect accuracy for CTR vs. EF group, whereas ANN and RF performed best for EF vs. non-EF group classification. Despite a slight decline in accuracy resulting from the incorporation of CTR samples, the overall discrimination remained high. We are fully aware that based on number and heterogeneity of patients involved in this study result transfer to clinical practice may have certain limitations. Given the explorative nature of this study the aim was to describe molecular mechanisms connecting BC TIME to the immunomodulatory treatment response in patients with HER2-positive BC. The above mentioned heterogeneity of patient cohort can be viewed as an advantage, as it allows ut to cover greater biological variability. Conclusions Our results demonstrate that modulation of ERS significantly influences BC cell viability, motility, and the antitumor activity of immune cells. ERS induction increased UPR markers (BiP, CHOP, XBP1s) in cancer and immune cells, while co-treatment with the chemical chaperone TUDCA at selected concentrations attenuated these effects – most effectively in PBMCs – indicating selective modulation of the immune component of TIME. These molecular changes translated into altered immune marker expression, and modified sensitivity to targeted therapies in in vitro co-culture models. Analyses of HER2-positive BC patient samples confirmed the clinical relevance of these findings: higher levels of TILs were found in non-EF patient samples, whereas UPR markers showed an opposite effect. These observations correlated with survival probability (EFS), where CD3, CD4, and CHOP carried the strongest predictive potential. Finally, we developed and validated a non-invasive predictive model using MALDI-TOF MS coupled with machine learning, which effectively distinguished treatment responders from non-responders. Together, these findings highlight ERS as a critical regulator of tumor–immune interactions and therapy efficacy in HER2-positive BC and support the use of mass-spectrometry-based biomarkers for personalized treatment strategies. In summary, our findings suggest that ERS modulation affects BC cell viability, motility, immune response, and therapy effectiveness, with prognostic implications in patients with HER2-positive BC . Moreover, MALDI-TOF MS combined with ML algorithms can effectively predict treatment response in patients with HER2-positive BC , potentially guiding personalized therapy decisions. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Masaryk Memorial Cancer Institute 2022/1680/MOU. Written informed consent was obtained from all the participants. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Consent for Publication All authors agree with the content of the manuscript. Funding Supported by the Ministry of Health of the Czech Republic, grant nr. NU21-03-00539. All rights reserved. Supported by the Ministry of Health of the Czech Republic, grant nr. NW24J-03-00038. All rights reserved. Supported by the SALVAGE project (OP JAC; reg. no. CZ.02.01.01/00/22_008/0004644) – co-funded by the European Union and by the State Budget of the Czech Republic. Supported by MH CZ - DRO (Masaryk Memorial Cancer Institute; 00209805). LK has been supported by the Grant Agency of Masaryk University (MUNI/C/0142/2023). Author Contribution BV and LM concieved and designed the study. LK prepared and carried out analyses. LP performed patient serum analysis using MALDI-TOF MS, data analysis, and designed a prediction model. BV conceptualized and drafted the manuscript. MU, LP, and LK performed statistical analyses. MH and IS developed the design of patient cohorts. All authors participated in the interpretation, and discussion of the results.BV and LK are both first authors, who contributed equally. Acknowledgement We acknowledge the core facility CELLIM supported by MEYS CR (LM2023050 Czech-BioImaging) and Biological Data Management and Analysis Core Facility funded by ELIXIR CZ research infrastructure (MEYS Grant No: LM2023055). All figures were created in Biorender. Patient samples were obtained by Biobank at Masaryk Memorial Cancer Institute, a coordinator of LRI BBMRI.cz; supported by the project BBMRI.cz no. LM2023033. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Panoff JE, et al. Risk of locoregional recurrence by receptor status in breast cancer patients receiving modern systemic therapy and post-mastectomy radiation. Breast Cancer Res Treat. 2011;128(3):899–906. Wolff AC, et al. 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Supplementary Files VavrusakovaKrejciSupplementarydatafinal.docx Suppl.Figure2.png Suppl.Figure1.png GraphicalabstractBCERS.jpeg Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 02 Feb, 2026 Submission checks completed at journal 01 Feb, 2026 First submitted to journal 27 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8709304","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584617020,"identity":"7013cf13-27aa-407e-81c9-1476f9fd518e","order_by":0,"name":"Barbora Vavrušáková","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Barbora","middleName":"","lastName":"Vavrušáková","suffix":""},{"id":584617024,"identity":"420f7c59-070a-4848-a073-40fc96fc021a","order_by":1,"name":"Lenka Krejčí","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Lenka","middleName":"","lastName":"Krejčí","suffix":""},{"id":584617026,"identity":"4590a4a1-8926-46e8-a229-160c80eadf74","order_by":2,"name":"Lukáš Pečinka","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Lukáš","middleName":"","lastName":"Pečinka","suffix":""},{"id":584617030,"identity":"1eb77e33-ef38-4468-b9fc-14fc61c9389f","order_by":3,"name":"Iveta Selingerová","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Iveta","middleName":"","lastName":"Selingerová","suffix":""},{"id":584617036,"identity":"8ca4644b-4e56-4adf-b19c-4b7974a9c7c2","order_by":4,"name":"Michal Uher","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Uher","suffix":""},{"id":584617038,"identity":"da3b2ab5-c8c3-48d0-892e-b415866e63fd","order_by":5,"name":"Miloš Holánek","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Miloš","middleName":"","lastName":"Holánek","suffix":""},{"id":584617040,"identity":"112f5207-9092-4492-87b3-04eaf053d0d2","order_by":6,"name":"Ema Říhová","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Ema","middleName":"","lastName":"Říhová","suffix":""},{"id":584617048,"identity":"c526f0ac-0fb3-4a4a-9656-0e0a25358d6a","order_by":7,"name":"Tomáš Kazda","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Kazda","suffix":""},{"id":584617050,"identity":"6f98bfbe-3b49-4e80-8897-388fc1b9bcb3","order_by":8,"name":"Lukáš Moráň","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACPgY2OJvxAVFa2JC0MBuQrIVNgjgtEmmJH38wbJMzbz97rOpGxWEG/v4DBLUcluZhuG0scyYv7XbOmcMMEgcIaklvkGZguJ04gyHH7HZu22EGA8YGglqaf/4AaeF/Y1YM1sJM2C/HJHhAWiRyzJjBWtgIaeF5lmbNY3DbWELijbF0zpl0HokzBLTws6cZ3/xRcVtOgj/H8HNOhbUcwRCDAKQ45CFG/SgYBaNgFIwCAgAAl3g4W0NI6ZUAAAAASUVORK5CYII=","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":true,"prefix":"","firstName":"Lukáš","middleName":"","lastName":"Moráň","suffix":""},{"id":584617055,"identity":"74ecdd54-bfa6-4634-ac6f-c8bf37171240","order_by":9,"name":"Marek Svoboda","email":"","orcid":"","institution":"Masaryk Memorial Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Marek","middleName":"","lastName":"Svoboda","suffix":""}],"badges":[],"createdAt":"2026-01-27 10:40:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8709304/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8709304/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101852229,"identity":"43b28f42-6c83-4e65-a1f5-192104e42135","added_by":"auto","created_at":"2026-02-04 10:11:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3692859,"visible":true,"origin":"","legend":"\u003cp\u003eERS arises when protein-folding homeostasis is disrupted, triggering the unfolded protein response (UPR) through the ER sensors IRE1α, PERK, and ATF6. The UPR initially aims to restore homeostasis by reducing protein synthesis, enhancing chaperone production, and activating autophagy and antioxidant responses. However, severe or persistent ERS can lead to apoptosis via CHOP, JNK, or caspase pathways. In cancer, cells exploit UPR signaling to promote survival, metabolic adaptation, autophagy, and resistance to apoptosis. ERS in tumor cells can also spread to neighboring cells—a process known as transmissible ERS (TERS)—which reprograms immune cells and suppresses antitumor immunity.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/cfe2e6a5b05dc2a68059d62b.png"},{"id":101852228,"identity":"37887595-9e35-44f4-a0bd-579264805c48","added_by":"auto","created_at":"2026-02-04 10:11:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8444533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003eanalysis of mono- and co-culture of SK-BR-3 and PBMCs treated with Tun and TUDCA and their combination.\u003c/p\u003e\n\u003cp\u003e(A) \u003cstrong\u003eCell viability of SK-BR-3 cells after 96 h of treatment\u003c/strong\u003e. Treatment with Tun led to a pronounced decrease in viability of SK-BR-3 cells, observed both in mono- and co-culture with PBMCs (p\u0026lt;0.05). No significant difference in viability was observed when comparing mono- and co-culture with PBMCs.\u003c/p\u003e\n\u003cp\u003e(B) \u003cstrong\u003eWound closure of SK-BR-3 cells 24 h after scratch\u003c/strong\u003e. Co-cultured cells exhibited slightly higher motility in control conditions (p\u0026lt;0.05), whereas under Tun treatment migration was significantly suppressed (p\u0026lt;0.05). In monoculture, no substantial differences in cellular motility were observed. Images were analyzed by automated mask-based measurement of the wound area over time (as seen in the representative image for each condition) and percent closure was calculated for each condition and normalized to control condition.\u003c/p\u003e\n\u003cp\u003e(C) \u003cstrong\u003eExpression analysis of SK-BR-3 cells\u003c/strong\u003e revealed strong upregulation of \u003cem\u003eHSPA5\u003c/em\u003e and \u003cem\u003eDDIT3\u003c/em\u003e after Tun treatment (p\u0026lt;0.05). Moderate increases were also seen in \u003cem\u003eERN1\u003c/em\u003e (p\u0026lt;0.05) and \u003cem\u003eXBP1s\u003c/em\u003e. TUDCA treatment partially suppressed this induced expression. In co-culture \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eACTA2\u003c/em\u003e expression increased markedly, and even more so when treated with Tun (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e(D) \u003cstrong\u003eIn PBMCs monoculture\u003c/strong\u003e, Tun exposure resulted in increased expression of \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3\u003c/em\u003e end \u003cem\u003eERN1\u003c/em\u003e(p\u0026lt;0.01), \u003cem\u003eATF6\u003c/em\u003e, and \u003cem\u003eBCL2\u003c/em\u003e (p\u0026lt;0.05), whereas combination with TUDCA reduced these expressions (exception \u003cem\u003eBCL2\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e(E) \u003cstrong\u003eHeat map visualization summarizes gene expression profiles\u003c/strong\u003e, which highlights strong upregulation of \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3,\u003c/em\u003e and \u003cem\u003eERN1\u003c/em\u003e genes under Tun treatment, and partial attenuation upon TUDCA co-treatment. This trend was even more visible in PBMCs monoculture in almost all UPR-related genes. EMT-associated genes \u003cem\u003eVIM\u003c/em\u003e, \u003cem\u003eACTA2,\u003c/em\u003e and pro-apoptotic marker \u003cem\u003eBCL2\u003c/em\u003e were upregulated in co-culture, and even more so with Tun treatment. The expression of \u003cem\u003eSNAI2\u003c/em\u003e, and in SK-BR-3 even \u003cem\u003eCDH2\u003c/em\u003e, was undetected in both cell types. Immune related genes were nearly unaltered.\u003c/p\u003e\n\u003cp\u003e(F) \u003cstrong\u003eAt the protein level\u003c/strong\u003e, Tun exposure increased BiP and CHOP, whereas co-treatment with TUDCA attenuated this effect. Tun also caused a decrease in the levels of immune proteins PD-L1, PD-1, and FOXP3.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/a844f3b61078f50431b61465.png"},{"id":101852195,"identity":"ad6b2130-5e04-4fa3-abef-2d1c8e8096e7","added_by":"auto","created_at":"2026-02-04 10:11:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6139908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining and analysis\u003c/strong\u003e of \u003cem\u003ein vitro\u003c/em\u003e mono- and co-culture of SK-BR-3.\u003c/p\u003e\n\u003cp\u003e(A)\u003cem\u003e \u003c/em\u003eERS markers BiP in green, CHOP in orange, cell nuclei-DAPI-in blue, and actin filaments-Phalloidin-in red. Scale bar 100 µm. BiP localized predominantly in the cytoplast and ER granules, while CHOP shifted from mainly cytoplasm to the nucleus upon Tun treatment.\u003c/p\u003e\n\u003cp\u003e(B) Masks from all fluorescence channels were combined to estimate the overall tissue area. \u0026nbsp;This mask was applied to all channels, and relative fluorescence intensity within the masked areas was measured. Tun slightly enhanced the intensity of BiP (top) and CHOP (bottom) signals in SK-BR-3 cells, with maximum intensity at T+T co-treatment in co-cultivation (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/fbae17abf4aebe38b2a23d7c.png"},{"id":101852107,"identity":"c4677cc9-5e64-4507-8a2a-2804d525e714","added_by":"auto","created_at":"2026-02-04 10:11:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6251026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003eanalysis of mono- and co-culture of SK-BR-3 and PBMCs treated with immunomodulatory therapy (Her and Tec) in combination with Tun and/or TUDCA.\u003c/p\u003e\n\u003cp\u003e(A) \u003cstrong\u003eCell viability of SK-BR-3 following 72 h of Her treatment combinations\u003c/strong\u003e. Her treatment alone led to a moderate reduction in SK-BR-3 cell viability, and even more so in co-culture conditions (p\u0026lt;0.05). A marked decline in viability under Tun exposure was observed (p\u0026lt;0.05). TUDCA alone showed no cytotoxic effect; on the contrary, it slightly improved viability in co-cultured conditions.\u003c/p\u003e\n\u003cp\u003e(B) \u003cstrong\u003eTec\u003c/strong\u003ealone caused only minimal change in viability of SK-BR-3 cells after 72 h of treatment. Exposure to Tun produced a slight decrease in viability, while TUDCA in co-culture again acted as a protective agent.\u003c/p\u003e\n\u003cp\u003e(C) \u003cstrong\u003eThe heat map provides a global overview of the gene expression changes across all conditions of Her treatment.\u003c/strong\u003e UPR associated genes, mainly \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3,\u003c/em\u003e and \u003cem\u003eERN1\u003c/em\u003e, as well as immune marker \u003cem\u003eIL2\u003c/em\u003e, were elevated after Tun treatment in co-cultivation, whereas co-treatment with TUDCA partially reduced this induction in SK-BR-3. \u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003eDDIT3\u003c/em\u003e were upregulated in co-culture conditions. In PBMCs the effect of treatment is similar, bud in larger magnitude than in SK-BR-3. \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eXBP1s\u003c/em\u003e, \u003cem\u003eBCL2,\u003c/em\u003e and \u003cem\u003eIFNG\u003c/em\u003e were increased nearly solely in PBMCs monoculture after Tun treatment.\u003c/p\u003e\n\u003cp\u003e(D) For the \u003cstrong\u003eTec\u003c/strong\u003earm, both cell types displayed patterns paralleling Her, but with slightly stronger upregulation in PBMCs, mainly in immune related genes (\u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eCD4\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e(E) \u003cstrong\u003eThe expression of most prominent genes\u003c/strong\u003e in SK-BR-3 cells (left) and PBMCs (right) treated with \u003cstrong\u003eHer combinations\u003c/strong\u003e. SK-BR-3 cells show only slight changes in expression in monoculture. The co-culture exposed to Tun shows increased \u003cem\u003eHSPA5\u003c/em\u003e(p\u0026lt;0.01), \u003cem\u003eDDIT3\u003c/em\u003e, \u003cem\u003eXBP1s\u003c/em\u003e (p\u0026lt;0.05), and \u003cem\u003eBAX\u003c/em\u003e. In co-culture conditions the overall expression of \u003cem\u003eBAX\u003c/em\u003e rises, whereas \u003cem\u003eXBP1s\u003c/em\u003e lowers (p\u0026lt;0.05). Expression of \u003cem\u003eCD8A \u003c/em\u003edecreases when treated with combination of Her + TUDCA + Tun in co-cultures (p\u0026lt;0.05)\u003cem\u003e.\u003c/em\u003e PBMCs respond strongly to Tun with pronounced upregulation of \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3\u003c/em\u003e, \u003cem\u003eATF6\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e (p\u0026lt;0.05), and \u003cem\u003eENTPD1\u003c/em\u003e, although this was mitigated by combination with TUDCA (p\u0026lt;0.05 for \u003cem\u003eATF6\u003c/em\u003eand \u003cem\u003eENTPD1\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e(F) \u003cstrong\u003eThe most significant transcriptional responses\u003c/strong\u003e in SK-BR-3 (left) and PBMCs (right) treated with \u003cstrong\u003eTec combinations\u003c/strong\u003e. In SK-BR-3 Tec + Tun led to a strong increase in \u003cem\u003eHSPA5\u003c/em\u003e (p\u0026lt;0.05) and \u003cem\u003eDDIT3\u003c/em\u003e, while TUDCA co-treatment further enhanced these levels. The expressions of \u003cem\u003eXBP1s\u003c/em\u003e (p\u0026lt;0.05) and \u003cem\u003eCD4\u003c/em\u003e also slightly raised with Tec + Tun treatment, whereas when TUDCA was added, these levels decreased (p\u0026lt;0.05 for CD4). In PBMCs Tec + Tun caused a global upregulation of the tested genes, particularly \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3\u003c/em\u003e, \u003cem\u003eERN1\u003c/em\u003e (p\u0026lt;0.05), and \u003cem\u003eCD4\u003c/em\u003e, while the co-treatment with TUDCA slightly alleviated this response (p\u0026lt;0.05 for \u003cem\u003eCD4\u003c/em\u003e), with exception of \u003cem\u003eERN1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(G) \u003cstrong\u003eAt the translational level\u003c/strong\u003e, the treatment with Her + Tun led to an increase in the levels of ERS proteins, particularly BiP, CHOP, XBP1s, and to a lesser extent IRE1α. BiP and XBP1s proteins were less abundant under co-culture conditions with Her + Tun treatment, whereas CHOP and IRE1α show a slight increase when compared with monoculture. HER2 protein levels were generally high and decreased slightly in the presence of PBMCs, and even more so with Her.\u003c/p\u003e\n\u003cp\u003eIn PBMCs the strongest signal of IRE1α and CHOP was observed under co-treatment Her + \u0026nbsp;TUDCA + Tun. Her + Tun treatment led to a decrease in immune related markers, mainly PD-L1, PD-1, CD3, and FOXP3. This effect was revoked to some extend with addition of TUDCA in PD-1 and CD3.\u003c/p\u003e\n\u003cp\u003e(H) In Tec-treated cultures, the protein expression profile was relatively similar but quantitatively less pronounced than with Her.\u003c/p\u003e\n\u003cp\u003eAcross all cell lines, Tun treatment was associated with the appearance of PD-L1 and PD-1 protein isoforms with lower molecular weight.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/0f3ae4a7eba33fb4f0821a62.png"},{"id":101852208,"identity":"7bba1d18-2f68-4727-94f2-aa79e533c09d","added_by":"auto","created_at":"2026-02-04 10:11:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15433233,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses based on tumor tissue and intraoperative serum samples from patients with HER2-positive BC treated with anti-HER2 therapy in combination with chemotherapy. Patients were divided according to the occurrence of EF – EF and non-EF group.\u003c/p\u003e\n\u003cp\u003e(A) Representative images of patient-derived tumor tissue immunofluorescence staining. Tissues were stained for UPR markers BiP and CHOP, immune markers CD3, CD4, and CD8 and cell nuclei (DAPI). Non-EF patients had higher TILs infiltration and lower levels of UPR markers. Spatial phenotyping was performed according to the workflow as described in section “\u003cem\u003eStatistical analyses\u003c/em\u003e”.\u003c/p\u003e\n\u003cp\u003e(B) EF patients showed higher relative intensity of signal of BiP and CHOP, and higher occurrence of CHOP in the vicinity of CD3+ TILs (CHOP immuno), whereas occurrence of CD4 was lower. Patients with lower probability of EFS had higher relative intensity of CHOP, higher levels of CHOP in the vicinity of CD3+ TILs, and conversely lower levels of CD4 (compared to median).\u003c/p\u003e\n\u003cp\u003e(C,D,E) (O)PLS-DA score plot of evaluated MALDI-TOF MS serum samples profiles (CTR – blue, non-EF – green, EF – red; 44 m/z signals, and average from 5 technical repetitions). Data allows to clearly separate between clinical groups.\u003c/p\u003e\n\u003cp\u003e(F,G,H) The accuracy of predictive models based on 5×repeated 5-fold CV (median, 95% confidence interval).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/e3221e4bb2945ab0718a5bf7.png"},{"id":101852277,"identity":"f222990b-4136-4208-9d2e-c90f856e778f","added_by":"auto","created_at":"2026-02-04 10:12:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36100885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/032ba248-9fa7-4a29-99a5-cdd95a9fff21.pdf"},{"id":101852239,"identity":"45cb5075-3ec7-4085-852f-78147736be59","added_by":"auto","created_at":"2026-02-04 10:11:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":926249,"visible":true,"origin":"","legend":"","description":"","filename":"VavrusakovaKrejciSupplementarydatafinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/97394b643a56ba68554c32f1.docx"},{"id":101852246,"identity":"fc4b7019-476b-4008-8346-2a5e66845b3c","added_by":"auto","created_at":"2026-02-04 10:11:35","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":774100,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/6075dc34cdedea5a1c851053.png"},{"id":101852108,"identity":"bbf44f48-8e34-49a7-9b91-abf3933d7eea","added_by":"auto","created_at":"2026-02-04 10:11:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1032116,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/4c575ea9450513e9c381ee9a.png"},{"id":101852245,"identity":"ad82d0b2-2a7a-4b76-acba-51097a0cd854","added_by":"auto","created_at":"2026-02-04 10:11:35","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1413071,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractBCERS.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8709304/v1/429298b723777cfe30e36d77.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunomodulatory Mechanisms of Endoplasmic Reticulum Stress in the Tumor Immune Microenvironment and Prediction of Treatment Response in HER2-Positive Breast Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHER2-positive breast cancer (BC) accounts for 15\u0026ndash;20% of all cases. The introduction of targeted therapies such as trastuzumab and pertuzumab has significantly improved patient outcomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. HER2-activated BCCs are more vulnerable to damage to the inhibition of HER2 signaling, particularly in context of radiotherapy. HER2 gene amplification/protein overexpression predicts poor prognosis and response to certain types of chemotherapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and correlates with a significantly shorter disease-free survival. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] \u003cb\u003eThe tumor immune microenvironment (TIME)\u003c/b\u003e contains non-transformed host cellular components of the tumor mass residing within the tumor region [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The BC TIME plays an important role in tumor development, progression and metastasis, anti-tumor immunity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], is highly plastic, and undergoes constant changes and stage-specific adaptations depending on various cancer cell-intrinsic and extrinsic factors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Throughout the progression of BC, the stroma undergoes significant changes, including infiltration of immune cells, inflammation, angiogenesis, and remodeling of the ECM [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within the local TIME, immune cells can acquire functional plasticity and adapt, which allows them to play dual role as either pro or anti-tumor agent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cancer cells employ various immune evasion strategies that can be enhanced by TIME and thus can suppress the infiltration, activation, and effector functions of immune cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Since TIME elements are involved in various stages of tumorigenesis, the TIME appears to be an attractive therapeutic target. Tumor-infiltrating lymphocytes (TILs) in TIME regulate the anti-tumor immunity, immunosuppression, immunotherapy effectiveness, invasiveness and metastasis, and chemo- and radiotherapy resistance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA variety of stress factors can impair endoplasmic reticulum (ER) homeostasis leading to the state of the \u003cb\u003eendoplasmic reticulum stress (ERS)\u003c/b\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Homeostasis disruption leads to the accumulation of abnormal proteins in the ER lumen, which sequester the ER-chaperone BiP. This triggers specific signaling pathways, collectively known as the unfolded protein response (UPR), which significantly influence cell fate. UPR consists of \u003cb\u003ethree main UPR signaling pathways\u003c/b\u003e, controlled by the proteins IRE1α, PERK, and ATF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These pathways help restore normal functions following cell injuries. \u003cb\u003eIRE1α\u003c/b\u003e allows to activate the \u003cem\u003eXBP1\u003c/em\u003e gene, which helps to restore protein balance by promoting the production of chaperone proteins and proteins involved in breaking down misfolded proteins [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cb\u003ePERK\u003c/b\u003e halts global protein synthesis by phosphorylating eIF2α. This reduces protein production but allows specific proteins like ATF4 to be made. ATF4 then triggers the production of proteins related to oxidative stress and autophagy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Lastly, \u003cb\u003eATF6\u003c/b\u003e promotes the transcription of genes involved in chaperone production, lipid metabolism, and UPR regulation, helping reduce ERS. In response to ERS, the UPR works to protect the cell by producing chaperone proteins, regulating lipid metabolism, and activating autophagy and antioxidant responses. However, depending on the extent of cellular damage, the UPR can activate either pro-survival or pro-apoptotic mechanisms. In case of persisting or severe ERS, the UPR can trigger cell death. This occurs through pathways like ATF4 and ATF6 activating the pro-apoptotic CHOP protein, IRE1α activating apoptosis via TRAF2 and JNK, or IRE1α activating caspases. There is abundant evidence that ERS-mediated response pathway is strictly associated with the pathogenesis of many human diseases including cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In healthy cells, UPR pathways aim to resolve cell injury or initiate cell death, depending on the severity of the damage and cellular/tissue status. However, \u003cb\u003ecancer cells often manipulate these stress mechanisms to support their survival\u003c/b\u003e by reprogramming their metabolism, activating oxidative stress responses, promoting autophagy, and inhibiting apoptosis and senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional studies have demonstrated that the tumoral UPR actively communicates with stromal cells, influencing cancer progression in a cell-nonautonomous manner. \u003cb\u003eTransmissible ERS (TERS)\u003c/b\u003e was initially described as a form of UPR-mediated intercellular communication between cancer cells and normal cell types [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies have shown that ER-stressed cancer cells are capable of activating the UPR in macrophages and other immune cells via the secretion of soluble factors into the culture media. This can lead to suppression of antitumor immune responses [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Cancer cells were shown to activate the UPR of surrounding cancer cells, enhancing resistance to standard anticancer drugs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUPR in immune cells plays a crucial immunosuppressive role within the TIME, supporting cancer progression, immune evasion, and therapy resistance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Tumor cells manipulate UPR pathways\u0026mdash;particularly PERK and IRE1α-XBP1 signaling\u0026mdash;to reprogram immune cells, leading to impaired antigen presentation, dysfunctional T cell responses, and diminished dendritic cell (DCs) activity [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This reprogramming also fosters immunosuppressive phenotypes in tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Targeting UPR-mediated immunomodulatory mechanisms offers promising avenues for novel cancer treatment strategies. Of note, the UPR has been targeted in noncancer cells (i. e., immune cells or whole body), observing important anticancer effects. These findings raise questions about the biological significance of TERS in cancer, emphasizing the need for further investigation to confirm the existence and impact of cell-to-cell UPR transmission under physiological conditions.\u003c/p\u003e \u003cp\u003eBlocking ERS sensors with small molecules can effectively suppress tumor growth by inhibiting angiogenesis, which was observed in various cancer types including BC [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Employing selective UPR inhibitors has the potential to enhance the function of TAMs and DCs, thereby mitigating the immunosuppressive environment within tumors. This strategy could be especially advantageous when combined with immune checkpoint inhibitors, potentially boosting their effectiveness. Supporting this idea, pharmacological inhibition of IRE1 and PERK signaling pathways in TAMs improved anti-PD-1 therapy efficacy by reducing the immunosuppressive phenotype of macrophages, lowering PD-L1 expression, promoting T cell activity and delayed tumor growth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTargeting BiP reduces CD47 expression, leading to increased tumor macrophage infiltration and reduced resistance to anti-estrogen therapy. Co-expression of BiP and CD47 correlates with poor prognosis in BC patients, highlighting crosstalk between the UPR and immune regulation in shaping therapeutic outcomes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study explores novel strategies to enhance the antitumor activity of immune cells by modulating ERS within the TIME, aiming to overcome immunosuppressive mechanisms and ultimately improve the effectiveness of anticancer therapies in HER2-positive BC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from healthy donor buffy coats by density gradient centrifugation and cultured in complete OpTmizer medium supplemented with FBS, L-glutamine, antibiotics, and IL-2. The HER2-positive breast cancer cell line SK-BR-3 was obtained from ATCC, authenticated by STR profiling, and maintained in high-glucose DMEM with FBS and antibiotics. For co-culture experiments, SK-BR-3 cells were seeded and allowed to adhere for 24 h, after which PHA-activated PBMCs were added at a 2:1 ratio. Co-cultures were treated with ERS modulators (TUDCA, tunicamycin), anti-HER2 trastuzumab, anti-PD-L1 atezolizumab, or their combinations at defined working concentrations.\u003c/p\u003e \u003cp\u003eCell viability was assessed by MTT assay, measuring formazan absorbance at 595 nm. Cell migration was evaluated using a scratch wound assay with live-cell imaging over 24 h. Gene expression was analyzed by RT-qPCR following phenol-chloroform RNA isolation, cDNA synthesis, and amplification using ACTB as a reference gene. Protein expression was examined by SDS-PAGE and Western blotting, with β-actin as a loading control. Immunofluorescence staining of in vitro co-cultures was performed to visualize target proteins, actin cytoskeleton, and nuclei using fluorescence microscopy.\u003c/p\u003e \u003cp\u003eRetrospective clinical analyses included HER2-positive breast cancer patients treated with anti-HER2 therapy plus chemotherapy, with ethical approval and informed consent. Patients were stratified by early failure, and survival outcomes were analyzed using Kaplan\u0026ndash;Meier estimates and log-rank tests. Tumor FFPE sections were processed for immunofluorescence microscopy. Serum proteomic profiling was conducted by MALDI-TOF MS following protein extraction, with spectra analyzed using multivariate statistical methods to identify discriminative features between patient groups and healthy controls.\u003c/p\u003e \u003cp\u003eFor extended version of Methods see Supplementary Information.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cstrong\u003eImpact of ERS modulation on BCCs viability and motility in vitro\u003c/strong\u003e \u003cp\u003eThe effects of ERS modulation within TIME on cancer and immune cells were evaluated in both mono- and co-cultures of PBMCs and BCCs treated with TUDCA, a chemical chaperone attenuating general ERS, and Tun, as an established inducer of ERS inhibiting the N-glycosylation machinery. Effective concentrations were established to ensure that TUDCA exerted its positive ERS modulating effect primarily on immune cells rather than tumor cells. Tun led to a distinct decrease in cell viability both in mono- and co-culture conditions. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). BC in co-culture exhibited slightly higher motility in control conditions, whereas under Tun, migration was slightly suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eERS modulation can drive gene and protein expression changes specifically in immune cells\u003c/strong\u003e \u003cp\u003eTo link the pilot observations with underlying molecular mechanisms, cells were treated for 24 h with selected concentrations of ERS modulators and treatment-associated gene expression alterations in UPR and immune markers were assessed. The effect of Tun in UPR activation was evident in monoculture and co-culture; Tun increased the expression of UPR markers, particularly the major regulator of the UPR chaperone HSPA5/BiP and the proapoptotic transcription factor DDIT3/CHOP. The effect of TUDCA was particularly pronounced in PBMCs, where UPR signaling was attenuated. Differences in expression were also observed between mono- and co-cultures in response to modulating conditions, suggesting that PBMCs were targeted specifically. Given the role of EMT in cancer, the levels of EMT markers were examined. In SK-BR-3, changes in gene expression indicate activation of EMT processes under co-culture conditions. The effect of ERS on immune function was evaluated in co-cultures of BCCs and PBMCs. In PBMCs monoculture, Tun led to a slight increase in \u003cem\u003eCD4\u003c/em\u003e transcription. To cover population variability, PBMCs were isolated from different donors for each biological replicate. Therefore, gene expression of \u003cem\u003eCD3E\u003c/em\u003e, \u003cem\u003eCD4\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eIL2RA\u003c/em\u003e, and \u003cem\u003eENTPD1\u003c/em\u003e varied between biological replicates, depending on the donor of PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTo describe these effects on protein level\u003c/b\u003e, immunoblotting was performed to visualize ERS associated proteins and immune markers of both PBMCs and BCCs after 48 h treatment with selected concentrations of ERS modulators. SK-BR-3 cells exhibited signs of UPR activation even under control conditions, with increases in BiP, CHOP, and IRE1α protein levels upon Tun treatment and further modulation in co-culture. Tun exposure also led to decreased levels of immune regulatory proteins PD-L1, PD-1, and FOXP3. In PBMCs monoculture, Tun treatment elevated BiP, CHOP, and IRE1α levels, which were subsequently reduced\u0026mdash;particularly BiP\u0026mdash;by TUDCA. Tun additionally upregulated CD8 protein levels while downregulating FOXP3. Notably, immune marker expression in PBMCs varied depending on the donor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTo monitor the spatial localization and abundance of BiP and CHOP\u003c/b\u003e in \u003cem\u003ein vitro\u003c/em\u003e co-cultures, we employed IF staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In SK-BR-3 cells, elevated levels of BiP and CHOP proteins were present in the control condition, corresponding to results from immunoblotting. Tun induced only a partial increase in BiP and CHOP protein levels according to image analysis, while addition of TUDCA in co-culture further enhanced this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, BiP was present only in the cytoplasm, whereas CHOP was localized in all conditions in both the cytoplasm and the nucleus, with a shift to the nucleus upon Tun treatment. The effect of ERS modulation was also evident in PBMCs, where we detected increased levels of CHOP protein, most abundantin the presence of Tun, while this effect was successfully reversed by treatment with TUDCA. BiP protein was almost undetectable in PBMCs using this method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eERS modulation impact on the effectiveness of targeted immunomodulatory therapy in vitro\u003c/strong\u003e \u003cp\u003eCurrent strategies for HER2-positive BC include treatment with monoclonal antibodies such as Her and Tec. To simulate immunomodulatory conditions, these agents were applied to SK-BR-3 cells in combination with ERS modulators. A significant reduction in tumor cell viability of SK-BR-3 was observed following treatment with Tun and its combinations with Her / Tec, with the most pronounced effects seen in co-culture with PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Combined treatment of Tun with Her or Tec increased gene expression of UPR and apoptotic markers(mainly \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eDDIT3\u003c/em\u003e, \u003cem\u003eERN1, XBP1s\u003c/em\u003e, and \u003cem\u003eBCL2\u003c/em\u003e) in PBMCs. However, transcriptional activation of UPR markers was attenuated by the treatment with TUDCA. Importantly, TUDCA did not markedly affect expression of these markers in SK-BR-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). At protein level, increased expression of BiP, CHOP, and XBP1s was detected in PBMCs after the co-treatment of Tun and Her/Tec, with levels reduced upon TUDCA addition (mainly with Tec). Similarly, these UPR markers were upregulated in tumor cells primarily in response to combined treatments. TUDCA moderately decreased these protein levels, particularly CHOP. Notably, HER2 protein levels were reduced following Her treatment, in the presence of PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). In terms of immune activation, combined treatments of Tun and immunomodulation resulted in increased transcription of immune-related genes \u003cem\u003eCTLA4\u003c/em\u003e, \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, \u003cem\u003eCD4\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e, and \u003cem\u003eENTPD1\u003c/em\u003e in PBMCs. TUDCA co-treatment reduced expression of \u003cem\u003eCTLA4\u003c/em\u003e, \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eCD4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). Combination of Tun and immunomodulatory treatment decreased protein levels of CD3, CD4, CD8, and FOXP3, with partial restoration observed upon TUDCA addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). Similarly to other PBMCs-associated results, immune responses varied among PBMCs donors. Collectively, these findings indicate that modulation of ERS significantly influences tumor and immune cell interaction \u003cem\u003ein vitro\u003c/em\u003e. Tun-induced ERS amplifies UPR markers and suppresses immune-related proteins, while TUDCA mitigates several of these effects, suggesting a regulatory role.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTo apply findings to the clinical setting, analyses were extended to a cohort of patients with HER2-positive BC.\u003c/b\u003e A total of 21 patients (median age 59 years, range 21\u0026ndash;76 years) were enrolled in this study. Of these, 11 (52%) were diagnosed with stage II and 10 (48%) with stage III disease, and all patients but one had nodal involvement. Eight patients underwent primary surgery, while the remaining 13 received neoadjuvant therapy. Anti-HER2 therapy consisted of trastuzumab alone in 14 (67%) patients and trastuzumab plus pertuzumab in the remainder. Overall, 70% of patients completed at least 48 weeks of anti-HER2 therapy. Early failure occurred in 9 patients, more frequently in stage III disease (67%), although the difference was not statistically significant. No other clinicopathological characteristics were associated with early failure.\u003c/p\u003e \u003cp\u003eDuring a median follow-up of 80.5 months, the 5-year OS rate was 75% (95% CI 58\u0026ndash;97%), with 42% in EF patients and 100% in non-EF (p\u0026thinsp;=\u0026thinsp;0.009). The 3-year and 5-year EFS rates of 57% (95% CI 39\u0026ndash;83%) and 47% (95% CI 30\u0026ndash;74%), respectively.\u003c/p\u003e \u003cp\u003eNon-EF patients showed lower occurrence and relative intensity of UPR markers, whereas immune related markers were measured at higher levels than in the EF group. These findings largely correlated with the probability of EFS in these patients, where CHOP, CD3 and CD4 markers showed the most promising predictive potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B, \u003cem\u003eSuppl. Figure\u0026nbsp;1)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnsupervised PCA based on MALDI-TOF MS data showed a distinct separation between the CTR and EF groups, with partial overlap of non-EF samples (\u003cem\u003eSuppl. Figure\u0026nbsp;2A\u003c/em\u003e). The supervised PLS-DA and OPLS-DA (for two clinical groups) performed on all samples confirmed these results, demonstrating clear separation of CTR and EF groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), partial separation of non-EF and EF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and distinct clustering of CTR and non-EF and EF groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). All selected ML prediction models except DT demonstrate the capacity to discriminate between CTR and EF groups with a high accuracy, reaching a maximum of 99%, CI: 98\u0026ndash;100% for PLS-DA algorithms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). For differentiation between EF and non-EF groups, RF-based models achieved the optimal performance, with a maximum accuracy of 81%, CI: 75\u0026ndash;87% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Including CTR samples slightly reduced the accuracy to 80%, CI: 75\u0026ndash;85% for the RF-based model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Two signals, detected at m/z 2863.4 and 8926.8 Da, significantly contributed to the RF-based model (\u003cem\u003eSuppl. Figure\u0026nbsp;2B-C\u003c/em\u003e). The 8926.8 Da signal enables discrimination between the EF and non-EF groups, whereas the 2863.4 Da signal distinguishes the CTR group from both patient groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTumor cells, through their aberrant growth and metabolism, create a particularly inhospitable environment characterized by various stress factors such as low pH, nutrient deprivation, and hypoxia. These factors contribute to the disruption of ER homeostasis in many cell types within TIME, leading to ERS and the activation of UPR signaling pathways. In most tumor cells, the continuous activation of the UPR facilitates malignant progression by promoting angiogenesis, proliferation, and tolerance to hypoxia. In contrast, excessive ERS in immune cells impairs their function, thereby weakening antitumor immunity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHER2-positive BC is characterized by elevated levels of TILs within TIME [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These TILs significantly contribute to the therapeutic efficacy of monoclonal anti-HER2 antibodies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the impairment of immune cell function due to excessive ERS is highly undesirable, as it limits their ability to target tumor cells. Here we determine whether modulating ERS in immune cells within the TIME could enhance their capacity to target tumor cells effectively.\u003c/p\u003e \u003cp\u003eThe interaction of PBMCs with BCCs induced changes in UPR signaling \u003cem\u003ein vitro\u003c/em\u003e, together with an increase in the expression of the anti-apoptotic gene \u003cem\u003eBCL2\u003c/em\u003e, particularly upon Tun treatment, reflecting stress adaptation and survival signaling [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results suggest resistance of BCCs to immune response and reduced apoptotic signalling preventing PBMCs from mitigating BC \u003cem\u003ein vitro.\u003c/em\u003e Additionally, SK-BR-3 cells exhibited an increase in the expression of the pro-apoptotic gene \u003cem\u003eBAX\u003c/em\u003e, mainly under Her treatments.\u003c/p\u003e \u003cp\u003eTun induced ERS and activated UPR signaling, profoundly reduced cell viability under all tested conditions and slightly suppressed cellular motility in co-culture conditions in SK-BR-3 cells. In these cells, Tun primarily influenced the \u003cem\u003eDDIT3\u003c/em\u003e gene, which induces apoptosis and is associated with longer patient survival when highly expressed [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In SK-BR-3 cells, continuous activation of the UPR signaling was observed even under control conditions. Additionally, these cells exhibited higher levels of HER2 protein. This suggests that the SK-BR-3 cells correspond to a more aggressive tumor type, strongly driven by the HER2 growth factor. The persistent activation of UPR signaling pathways may enable these BCCs to better adapt to changing conditions and leverage this adaptability to their advantage.\u003c/p\u003e \u003cp\u003eWhen monitoring the effect of ERS modulators on the immune response to BCCs, the biological variability of individual PBMCs donors was highly evident, especially on the immune profile. TUDCA consistently attenuated UPR signaling in PBMCs, primarily reflected in decreased levels of HSPA5/BiP, \u003cem\u003eDDIT3\u003c/em\u003e/CHOP, \u003cem\u003eERN1\u003c/em\u003e/IRE1α, and \u003cem\u003eXBP1s\u003c/em\u003e/XBP1s. Notably, elevated levels of these proteins in TILs are typically associated with diminished infiltration into the TIME, disease progression, metastasis, and poorer clinical outcomes in patients with ovarian cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn SK-BR-3 cells, TUDCA partially suppressed Tun-induced UPR marker expression, with strongest attenuation in co-culture, suggesting that the apparent decrease in total protein levels originates from PBMCs rather than from BCCs. Furthermore, TUDCA led to reduced T-cell activation and regulatory markers in PBMCs, most prominently \u003cem\u003eFOXP3\u003c/em\u003e and \u003cem\u003eCD4\u003c/em\u003e, as well as a partial reduction in \u003cem\u003eCTLA4\u003c/em\u003e, \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eENTPD1\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis work also investigated the potential effect of the drug TUDCA in promoting the efficacy of anti-HER2 therapy. TUDCA in combination with the drug Her caused an even more pronounced attenuation of ERS signalling in PBMCs, particularly decreasing \u003cem\u003eHSPA5, DDIT3\u003c/em\u003e, and \u003cem\u003eERN1\u003c/em\u003e expression, thereby limiting pro-apoptotic UPR activation.\u003c/p\u003e \u003cp\u003eTUDCA reduced Tun-induced transcription of \u003cem\u003eIL2\u003c/em\u003e in both mono- and co-culture settings (predominantly produced by CD4\u0026thinsp;+\u0026thinsp;TILs in response to antigenic stimulation). IL2 promotes TILs proliferation and differentiation, and its administration promotes disease remission in some cancer patients [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the Tec arm, TUDCA moderately reduced overall UPR marker expression in both BCCs and PBMCs, but did not significantly enhance immune activation. Not only the abundance of individual stress markers but also their localization, suggestive of their characteristic activity, was examined. The localization of the UPR proteins BiP and CHOP was specific. BiP was present in all conditions only in the cytoplasm, specifically as granules in the ER [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, CHOP protein was translocated from the cytoplasm to the nucleus upon ERS, where it functions as a pro-apoptotic transcription factor, and this translocation is thus indicative of active signaling [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. CHOP remained partly nuclear even under control conditions, supporting persistent UPR activation in SK-BR-3 cells.\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that modulation of ERS significantly influences tumor and immune cell responses to targeted immunomodulatory therapy \u003cem\u003ein vitro\u003c/em\u003e. Tun promotes UPR activation and suppresses immune-related proteins, while TUDCA mitigates several of these effects, suggesting a regulatory role. Her reduced HER2 protein levels, particularly in co-culture conditions.\u003c/p\u003e \u003cp\u003eWe analyzed the molecular background of retrospectively obtained HER2-positive BC tumor tissue samples to identify the role of ERS in BC TIME and its\u0026rsquo; effect on infiltrated immune cells. We observed a positive correlation between EF and lower TILs infiltration (CD3+, CD4+, and CD8+) in the TIME. The number of TILs in tumor tissue was also correlated with treatment outcome by \u003cem\u003eRathore et al.\u003c/em\u003e, who described that infiltration of CD3+, CD4+, and CD8\u0026thinsp;+\u0026thinsp;TILs was significantly associated with better prognosis of BC patients and reduced risk of disease relapse [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similarly, we proved the level of CD4\u0026thinsp;+\u0026thinsp;TILs to be a good prognostic marker of overall EFS in the performed analysis. In this study, we also correlated the level of UPR markers in TIME with EFS. A higher BiP and CHOP signal in whole tissue, as well as in the vicinity of CD3\u0026thinsp;+\u0026thinsp;TILs, was observed in EF patients compared to the non-EF group. We can summarize that longer EFS is associated with more TILs in the TIME, their higher activity against BCCs and lower UPR activation in the TIME.\u003c/p\u003e \u003cp\u003eFinally, we developed predictive model based on MALDI-TOF MS analysis of peripheral blood serum. We showed that spectral data combined with PCA and supervised PLS/OPLS-DA effectively separated CTR and EF groups, while non-EF group displayed partial overlap with EF group. ML predictive models, particularly RF, k-NN, ANN, and PLS-DA, achieved near-perfect accuracy for CTR vs. EF group, whereas ANN and RF performed best for EF vs. non-EF group classification. Despite a slight decline in accuracy resulting from the incorporation of CTR samples, the overall discrimination remained high.\u003c/p\u003e \u003cp\u003eWe are fully aware that based on number and heterogeneity of patients involved in this study result transfer to clinical practice may have certain limitations. Given the explorative nature of this study the aim was to describe molecular mechanisms connecting BC TIME to the immunomodulatory treatment response in patients with HER2-positive BC. The above mentioned heterogeneity of patient cohort can be viewed as an advantage, as it allows ut to cover greater biological variability.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results demonstrate that modulation of ERS significantly influences BC cell viability, motility, and the antitumor activity of immune cells. ERS induction increased UPR markers (BiP, CHOP, XBP1s) in cancer and immune cells, while co-treatment with the chemical chaperone TUDCA at selected concentrations attenuated these effects \u0026ndash; most effectively in PBMCs \u0026ndash; indicating selective modulation of the immune component of TIME. These molecular changes translated into altered immune marker expression, and modified sensitivity to targeted therapies in \u003cem\u003ein vitro\u003c/em\u003e co-culture models.\u003c/p\u003e \u003cp\u003eAnalyses of HER2-positive BC patient samples confirmed the clinical relevance of these findings: higher levels of TILs were found in non-EF patient samples, whereas UPR markers showed an opposite effect. These observations correlated with survival probability (EFS), where CD3, CD4, and CHOP carried the strongest predictive potential.\u003c/p\u003e \u003cp\u003eFinally, we developed and validated a non-invasive predictive model using MALDI-TOF MS coupled with machine learning, which effectively distinguished treatment responders from non-responders.\u003c/p\u003e \u003cp\u003eTogether, these findings highlight ERS as a critical regulator of tumor\u0026ndash;immune interactions and therapy efficacy in HER2-positive BC and support the use of mass-spectrometry-based biomarkers for personalized treatment strategies.\u003c/p\u003e \u003cp\u003eIn summary, our findings suggest that \u003cb\u003eERS modulation affects BC cell viability, motility, immune response, and therapy effectiveness, with prognostic implications in patients with HER2-positive BC\u003c/b\u003e. Moreover, \u003cb\u003eMALDI-TOF MS combined with ML algorithms can effectively predict treatment response in patients with HER2-positive BC\u003c/b\u003e, potentially guiding personalized therapy decisions.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e This study was approved by the Ethics Committee of Masaryk Memorial Cancer Institute 2022/1680/MOU. Written informed consent was obtained from all the participants.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for Publication\u003c/h2\u003e \u003cp\u003eAll authors agree with the content of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eSupported by the Ministry of Health of the Czech Republic, grant nr. NU21-03-00539. All rights reserved. Supported by the Ministry of Health of the Czech Republic, grant nr. NW24J-03-00038. All rights reserved. Supported by the SALVAGE project (OP JAC; reg. no. CZ.02.01.01/00/22_008/0004644) \u0026ndash; co-funded by the European Union and by the State Budget of the Czech Republic. Supported by MH CZ - DRO (Masaryk Memorial Cancer Institute; 00209805). LK has been supported by the Grant Agency of Masaryk University (MUNI/C/0142/2023).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBV and LM concieved and designed the study. LK prepared and carried out analyses. LP performed patient serum analysis using MALDI-TOF MS, data analysis, and designed a prediction model. BV conceptualized and drafted the manuscript. MU, LP, and LK performed statistical analyses. MH and IS developed the design of patient cohorts. All authors participated in the interpretation, and discussion of the results.BV and LK are both first authors, who contributed equally.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the core facility CELLIM supported by MEYS CR (LM2023050 Czech-BioImaging) and Biological Data Management and Analysis Core Facility funded by ELIXIR CZ research infrastructure (MEYS Grant No: LM2023055). All figures were created in Biorender. Patient samples were obtained by Biobank at Masaryk Memorial Cancer Institute, a coordinator of LRI BBMRI.cz; supported by the project BBMRI.cz no. LM2023033.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePanoff JE, et al. Risk of locoregional recurrence by receptor status in breast cancer patients receiving modern systemic therapy and post-mastectomy radiation. Breast Cancer Res Treat. 2011;128(3):899\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolff AC, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. 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Indian J Med Res. 2014;140(3):361\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-mammary-gland-biology-and-neoplasia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jomg","sideBox":"Learn more about [Journal of Mammary Gland Biology and Neoplasia](http://link.springer.com/journal/10911)","snPcode":"10911","submissionUrl":"https://submission.nature.com/new-submission/10911/3","title":"Journal of Mammary Gland Biology and Neoplasia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HER2-positive Breast Cancer, Endoplasmic Reticulum Stress, Unfolded Protein Response, Tumor Immune Microenvironment, MALDI TOF Mass Spectrometry","lastPublishedDoi":"10.21203/rs.3.rs-8709304/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8709304/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHER2-positive breast cancer (BC) is an aggressive subtype with poor long-term outcomes in advanced disease, largely due to resistance to HER2-targeted therapies. Within the tumor-immune microenvironment (TIME), tumor and immune cells face diverse stressors, including endoplasmic reticulum stress (ERS). ERS activates the Unfolded Protein Response (UPR), which aims to restore homeostasis or trigger apoptosis. In BC cells, chronic UPR activation promotes malignant progression, while in immune cells ERS impairs function and weakens antitumor immunity. Given the relatively high immune infiltration in HER2-positive BC, preserving immune competence is crucial for therapeutic efficacy. This study aims to elucidate TIME-associated molecular mechanisms in HER2-positive BC using \u003cem\u003ein-vitro\u003c/em\u003e model and patient tumor samples analysis from 21 patients and to develop predictive models of treatment response using liquid biopsy-based mass spectrometry (MS) approach (21 patients, 15 healthy donors). ERS modulation was shown to significantly affect cancer\u0026ndash;immune cell interactions in co-culture models. Key ERS-related genes were characterized at the transcriptional, translational, and spatial levels. These findings were used to model the effects of immunomodulatory therapies \u003cem\u003ein vitro\u003c/em\u003e. Analysis of patient samples revealed distinct TIME patterns associated with treatment response. Serum-based MALDI-TOF MS enabled the development of a predictive model that discriminated responders from non-responders. Modulating ERS within the TIME represents a promising strategy to enhance immune-mediated antitumor activity in HER2-positive BC. This study provides a detailed molecular characterization of the HER2-positive BC TIME and presents an MS- and machine learning-based predictive model for patient stratification.\u003c/p\u003e","manuscriptTitle":"Immunomodulatory Mechanisms of Endoplasmic Reticulum Stress in the Tumor Immune Microenvironment and Prediction of Treatment Response in HER2-Positive Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 10:09:33","doi":"10.21203/rs.3.rs-8709304/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-02-02T21:37:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-02T21:11:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-02T02:51:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Mammary Gland Biology and Neoplasia","date":"2026-01-27T10:11:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-mammary-gland-biology-and-neoplasia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jomg","sideBox":"Learn more about [Journal of Mammary Gland Biology and Neoplasia](http://link.springer.com/journal/10911)","snPcode":"10911","submissionUrl":"https://submission.nature.com/new-submission/10911/3","title":"Journal of Mammary Gland Biology and Neoplasia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3c4575de-8c4d-42d6-b2f6-d7191b87aa32","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T10:09:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 10:09:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8709304","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8709304","identity":"rs-8709304","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}