Collagen modification remodels the sarcoma tumor microenvironment and promotes resistance to immune checkpoint inhibition

Collagen modification remodels the sarcoma tumor microenvironment and promotes resistance to immune checkpoint inhibition | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Collagen modification remodels the sarcoma tumor microenvironment and promotes resistance to immune checkpoint inhibition Hehai Pan , Ying Liu , Ashley M. Fuller , Erik F. Williams , Joseph A. Fraietta , T.S. Karin Eisinger doi: https://doi.org/10.1101/2024.06.28.601055 Hehai Pan 1 Department of Pathology & Laboratory Medicine 2 Penn Sarcoma Program 3 Abramson Family Cancer Research Institute 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ying Liu 1 Department of Pathology & Laboratory Medicine 2 Penn Sarcoma Program 3 Abramson Family Cancer Research Institute 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashley M. Fuller 1 Department of Pathology & Laboratory Medicine 2 Penn Sarcoma Program 3 Abramson Family Cancer Research Institute 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site Erik F. Williams 4 Department of Microbiology 5 Center for Cellular Immunotherapies 6 Parker Institute for Cancer Immunotherapy 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joseph A. Fraietta 4 Department of Microbiology 5 Center for Cellular Immunotherapies 6 Parker Institute for Cancer Immunotherapy 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site T.S. Karin Eisinger 1 Department of Pathology & Laboratory Medicine 2 Penn Sarcoma Program 3 Abramson Family Cancer Research Institute 7 Perelman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: tsk.eisinger{at}gmail.com Abstract Full Text Info/History Metrics Preview PDF Abstract Molecular mechanisms underlying immune checkpoint inhibitor (ICI) response heterogeneity in solid tumors, including soft tissue sarcomas (STS), remain poorly understood. Herein, we demonstrate that the collagen-modifying enzyme, procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (Plod2), which is over-expressed in many tumors relative to normal tissues, promotes immune evasion in undifferentiated pleomorphic sarcoma (UPS), a relatively common and aggressive STS subtype. This finding is consistent with our earlier observation that Plod2 promotes tumor metastasis in UPS, and its enzymatic target, collagen type VI (ColVI), enhances CD8+ T cell dysfunction. We determined that genetic and pharmacologic inhibition of Plod2 with the pan-Plod transcriptional inhibitor minoxidil, reduces UPS growth in an immune competent syngeneic transplant system and enhances the efficacy of anti-Pd1 therapy. These findings suggest that PLOD2 is an actionable cancer target and its modulation could augment immunotherapy responses in patients with UPS, and potentially other sarcomas and carcinomas. Introduction Immunosuppression in the solid tumor microenvironment (TME) arises from mechanisms driven by both the tumor parenchyma and surrounding stroma 1 . One mechanism of particular clinical significance is tumor-induced activation of inhibitory immune checkpoints, mediated by co-receptors expressed on the surfaces of CD8 + T cells that attenuate their tumoricidal activity 2 , 3 . Accordingly, immune checkpoint inhibitors (ICIs) such as Pembrolizumab (a-programmed cell death protein 1, PD-1) are promising anti-neoplastic agents. However, clinical response rates vary widely (15-60%) among solid tumor patients, with heterogeneous responses reported even within specific cancer types 4 . Thus, a better understanding of ICI response heterogeneity is critical for the development of novel approaches that improve therapeutic efficacy. Soft tissue sarcoma (STS) is a large, heterogeneous group of solid connective tissue tumors that account for ∼1% of adult cancer cases (up to 350,000 cases worldwide annually) 5 , 6 . Undifferentiated pleomorphic sarcoma (UPS) is a relatively common STS subtype that predominantly arises in adult skeletal muscle 7 , 8 . Although STS is generally considered non-immunogenic due to its low mutational and neoantigen burden relative to carcinomas, recent clinical trials have revealed that ∼25% of UPS patients exhibit objective clinical responses to a-PD-1 treatment. 9 , 10 These encouraging findings suggest that studies of UPS may provide valuable insights into strategies for enhancing CD8 + T cell function and ICI responses in solid tumors. Defining characteristics of UPS, as well as other sarcomas and high-grade carcinomas, include extensive deposition and aberrant post-translational modification (PTM) of extracellular matrix (ECM) proteins 11 - 14 . In particular, the solid tumor ECM is rich in molecules belonging to the collagen superfamily, a large, diverse protein family that contains 28 molecular species encoded by over 40 genes 15 . Although the roles of individual collagen proteins in cancer-associated processes are only beginning to be defined - particularly with respect to their impact on solid tumor immune evasion - our recent work demonstrated that collagen type VI (ColVI), a microfibrillar collagen, facilitates tumor progression by inhibiting T cell migration, infiltration, and function 16 , 17 . Specifically, ColVI promotes immune evasion and hinders ICI efficacy in UPS by inactivating CD8 + T cells and remodeling collagen type I (ColI) in the TME 16 . These findings implicate ColVI ablation, in combination with immunotherapy, as a promising approach to mitigate immune evasion in UPS, and potentially other solid tumors. As ColVI molecules in the UPS TME are unlikely to be directly “druggable”, investigation of ColVI-interacting proteins may reveal potential ColVI-targeting strategies. The lysine hydroxylase procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) is a collagen-modifying enzyme required for normal collagen production across many tissue types; germline PLOD2 mutations result in Bruck syndrome, a rare, autosomal-recessive disease in which patients present with osteoporosis, scoliosis, and joint contractures due to under-hydroxylated COLI molecules 18 , 19 . In malignant contexts, we and others have established that PLOD2 overexpression promotes metastasis and reduces long-term survival in the setting of UPS and various epithelial tumors (e.g., bladder, liver, renal, and lung), in part through aberrant hydroxylation of ColVI in the TME 20 - 28 . However, because these prior in vivo studies were predominantly carried out in immunodeficient xenograft models, the role of cancer cell-intrinsic Plod2 in primary tumor immune evasion remains underexplored. Therefore, herein, we queried the role of UPS cell-intrinsic Plod2 in immunosuppression and adaptive immune cell function in the UPS TME. Our findings suggest targeting collagen modification as a novel approach to potentiate ICI efficacy in UPS, and potentially other solid tumors. Methods Cell lines Human STS-109 cells were derived from a pre-treatment human UPS tumor by Rebecca Gladdy, M.D. (University of Toronto) and cultured in DMEM with 20% (vol/vol) FBS, 1% L-glutamine, and 1% penicillin/streptomycin. HEK-293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM with 10% (vol/vol) FBS, 1% L-glutamine, and 1% penicillin/streptomycin. Murine sarcoma SKPY42.1 cells on a C57BL/6 background (a gift from Sandra Ryeom, PhD, Columbia University) were cultured in DMEM with 10% (vol/vol) FBS, 1% L-glutamine, and 1% penicillin/streptomycin. All cell lines were cultured at 37°C in 5% CO 2 and confirmed to be negative for mycoplasma contamination. Murine models All animal experiments were performed in accordance with NIH guidelines and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. We generated genetically engineered mouse model (GEMM) tumors by injecting a calcium phosphate precipitate of adenovirus expressing Cre recombinase (University of Iowa) into the right gastrocnemius muscle of 3-month-old LSL- Kras G12D+ ; Trp53 fl/fl (KP) mice as previously described 29 . For syngeneic transplant studies, 1 million SKPY42.1 cells were resuspended in 100 ul PBS and implanted subcutaneously into syngeneic 5-6-week-old C57BL/6 mice (The Jackson Laboratory; strain code 000664). In vivo drug treatment, tumor growth measurements, and survival analyses For minoxidil-alone studies, minoxidil (Sigma-Aldrich, M4145) or vehicle control (PBS) was administered intraperitoneally (I.P.) daily at 10 mg/kg or 30 mg/kg, beginning 12 days after initial implantation of UPS cells. In immune checkpoint studies, 200 μg of anti-Pd1 monoclonal blocking antibody (BE0146, BioXCell) or isotype control antibody (BE0089, BioXCell) was administered I.P. every three days once tumors became palpable. Minoxidil (or vehicle control) was administered at 30 mg/kg in all studies evaluating efficacy in combination with anti-Pd1 antibodies. In all studies, tumors were measured every 2-3 days using calipers, and volumes were calculated using the formula (ab 2 )π/6, where a and b indicate the longest and shortest dimensions, respectively. Body weights were recorded every 2-3 days, and tumor volumes of 2000 mm 3 were used as endpoints for survival analysis. Two photon second-harmonic generation (SHG) imaging of tumor collagen fibers Organization of collagen fibers in formalin-fixed, paraffin-embedded murine tumor sections were visualized with second harmonic generation (SHG) imaging using a Leica TCS SP8 MP 2-photon microscope (Leica Microsystems) as previously described 16 . The unstained tissue slides were maintained in water, and image stacks (6.99 μm) were acquired using a 25X 1.0NA water immersion objective with 4x zoom. Collagen fiber width and orientation distribution from SHG image stacks (maximum-intensity projections) were quantified using CT-FIRE as described previously 16 , 30 . RNA isolation and RT-qPCR Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Cat #74104) and tissue using TRIzol (Thermo Scientific, #15596018). Reverse transcription of mRNA was performed using the High-Capacity RNA-to-cDNA Kit (Thermo Scientific, #4387406). RT-qPCR was performed using a ViiA7 real-time PCR system (Applied Biosystems). All probes were TaqMan “Best coverage” probes (Life Technologies). HPRT1 was used as a normalization control. Lentiviral Transduction Glycerol stocks for human PLOD2 -targeting (TRCN0000064809, TRCN0000064808), murine Plod2 -targeting (TRCN0000076409, TRCN0000076411), and scrambled shRNAs were obtained from Sigma Aldrich. shRNA plasmids were packaged using the third-generation lenti-vector system (pMDLg/pRRE, pRSV-Rev, and pMD2.G/VSVG) and expressed in HEK-293T cells. Supernatant was collected at 24 and 48 hrs after transfection and subsequently concentrated using polyethylene glycol-8000. Virus transduction of target sarcoma cells was performed in the presence of 8.0 μg/mL polybrene (Sigma-Aldrich, #H9268) and puromycin selection (3 μg/mL) was performed after 48 hours. RT-qPCR was performed to confirm knockdown efficiency of target genes at day 5 after initial viral transduction. Human CAR-T cell production and xCELLigence real-time cytotoxicity assay The xCELLigence real-time cytotoxicity assay was performed as previously described. 16 In brief, STS109 cells (target cells) expressing a control or one of multiple PLOD2 -targeting shRNAs were harvested and seeded in triplicate into 96-well polyethylene terephthalate plates (E-Plate VIEW 96 PET, Agilent) at 20,000 cells/per well. STS-109 cells were allowed to adhere to the bottoms of wells for 24 hours (37°C, 5% CO 2 conditions) after which effector cells (CART-TnMUC1 cells or donor-matched un-transduced T cells [NTD cells]) were added to wells at effector:target cell ratios of 10:1, 5:1, 1:1, and 0:1. Cytotoxic activity of the T lymphocytes was determined via continuous acquisition of impedance data over 6 days. Raw impedance data was analyzed using 762 RTCA 2.1.0 software as described in 16 . Human samples De-identified human sarcoma and non-malignant muscle samples were obtained from surgically resected tumors from patients undergoing therapeutic surgical resection in accordance with protocols approved by the Institutional Review Board at the University of Pennsylvania. Computational analyses of human samples Associations between PLOD2 expression levels and human patient survival in The Cancer Genome Atlas-Sarcoma (TCGA-SARC) dataset were queried with the cBioPortal web server ( https://www.cbioportal.org/ ). The publicly available Affymetrix Human Genome U133A microarray dataset reported in Detwiller et al. 31 ; NCBI Gene Expression Omnibus accession number GSE2719) was used to compare PLOD2 gene expression levels in sarcoma and normal connective tissue. Data pre-processing was carried out in RStudio as follows: Data were quantile normalized, and duplicate probes corresponding to the same ENTREZ gene ID were collapsed by averaging. The resulting gene expression values were log-transformed with a pseudocount of 1 and median centered across samples. Statistical analysis Statistical analysis was performed using GraphPad Prism (version 10). Data are shown as mean ± SEM or SD as indicated in the figure legends. Student t-tests (unpaired two-tailed) were performed to determine whether the difference between two means was statistically significant. ANOVA was used for such assays with three or more groups. 2-way repeated-measures ANOVA, mixed-effects models, or non-linear regression models (exponential fit) were used for analyses of in vivo tumor growth curves. Results Plod2 promotes UPS primary tumor growth in an immunocompetent setting Previous reports investigating the role of cancer cell-intrinsic Plod2 in primary tumor growth have primarily relied on xenograft studies in nude mice. These animals lack mature adaptive immune cells (T cells and B cells) but generally retain innate immunity (e.g., myeloid-lineage cells). Therefore, to understand the role of UPS cell-intrinsic Plod2 in immune evasion, we leveraged the Kras G12D/+ ; Trp53 fl/fl (KP) syngeneic transplant system previously introduced in 16 . In this system, sarcoma cells derived from the gold-standard KP genetically engineered mouse model (GEMM) of UPS 29 on a pure C57BL/6 background (herein referred to as “KP cells”) are implanted subcutaneously or orthotopically (intramuscularly) into syngeneic, immunocompetent C57BL/6 mice. 16 To this end, we transduced KP cells with a control (shScr) or one of multiple Plod2- targeting (shPlod2) shRNAs, injected them subcutaneously into recipient animals, and tracked tumor growth ( Fig. 1A-C ). Remarkably, unlike in previous work where UPS cell-intrinsic Plod2 depletion had no impact on primary tumor progression in immunodeficient nude mice 20 , Plod2 -deficient UPS tumors were substantially smaller than Plod2- sufficient controls in an immunocompetent setting. To validate this genetic observation with a pharmacologic approach, we treated syngeneic KP UPS tumor-bearing mice with minoxidil, a non-specific transcriptional inhibitor of Plod2 and its homologs Plod1 and Plod3 32 ; Fig. 1D ). Minoxidil induced modest dose-dependent reductions in UPS tumor growth, confirming that Plod2 depletion can suppress primary tumor progression in the context of an intact immune system. Together, these results suggest that the presence of adaptive immune cells is required for Plod2-dependent UPS tumor growth, and that cancer cells expressing high levels of Plod2 may promote immunosuppression. Download figure Open in new tab Figure 1: UPS cell-intrinsic Plod2 promotes primary tumor growth in an immunocompetent setting. A . Validation of Plod2 expression in KP UPS cells (SKPY42.1 cell line) prior to in vivo implantation. Mean + SD. Technical replicates; no statistics are shown. B . Tumor growth curves from subcutaneous syngeneic transplant of 1 × 10 6 KP cells from A in C57BL/6 mice. Not significant by two-way repeated measures ANOVA with Dunnett’s post-hoc test (vs. shScr). C . Weights of excised tumors from B . One-way ANOVA with Dunnett’s post-hoc test (vs. shScr). D . Tumor growth curves of subcutaneous syngeneic KP tumor-bearing mice treated daily (i.p.) with 10 mg/kg or 30 mg/kg minoxidil, beginning 12 days after UPS cell implantation. Not significant (mixed-effects model with Dunnett’s post-hoc test vs. shScr). For B-D , error bars indicate mean + SEM. Plod2 inhibition potentiates immune checkpoint inhibitor efficacy in UPS To further investigate the relationship between UPS cell-intrinsic PLOD2 and suppression of adaptive immunity, we explored the effects of PLOD2 + UPS cells on T cell function. We focused on T cells in these assays, rather than B cells, because T cells are critical players in endogenous anti-tumor immune responses and key targets of immunotherapy strategies 33 . First, we leveraged Tn-MUC1 chimeric antigen receptor T cells (Tn-MUC1 CAR T cells), which target the Tn isoform of mucin 1, a cancer neoantigen 34 . We co-cultured these cells with Tn-MUC1 + primary human STS-109 UPS cells 16 expressing a control or one of multiple PLOD2- targeting shRNAs at multiple effector:target ratios, and tracked longitudinal UPS cell lysis ( Figure 2A-B ). This experiment revealed that T cell cytolysis was enhanced in the presence of PLOD2- deficient UPS cells, confirming that UPS cells expressing high levels of PLOD2 suppress T cell function. To explore this relationship in vivo , we treated mice bearing syngeneic subcutaneous KP tumors with minoxidil, alone or in combination with ɑ-Pd1 checkpoint therapy. We hypothesized that minoxidil would augment the efficacy of immune checkpoint blockade due to enhanced T cell function in the setting of attenuated UPS cell-intrinsic Plod2 expression. Consistent with this hypothesis, combination treatment with minoxidil and ɑ-Pd1 significantly impaired primary UPS tumor progression compared to treatment with either monotherapy alone ( Figure 2C ). We confirmed this observation in the gold-standard autochthonous KP GEMM, in which mice receiving combination therapy (minoxidil + ɑ-Pd1) exhibited increased survival times (time-to-maximum tumor volume) compared to animals receiving single-agent treatments ( Figure 2D ). Thus, we conclude that pharmacologic Plod2 suppression potentiates the efficacy of ɑ-Pd1 checkpoint therapy in UPS. Download figure Open in new tab Figure 2: Pharmacologic inhibition of Plod2 potentiates the efficacy of ɑ-Pd1 checkpoint therapy in UPS. A . Representative longitudinal cytolysis curves of shScr or shPLOD2 human STS-109 UPS cells co-cultured with CART-TnMUC1 cells from 1 independent human donor. Measurements indicate percent target (UPS) cell cytolysis. B . Quantification of data from A . Statistics are not shown because n < 3 (n = 2). C . Growth curves of subcutaneous (flank) syngeneic tumors in C57BL/6 mice treated with minoxidil (or vehicle control), alone or in combination with ɑ-Pd1 (or isotype control) antibodies. Growth curves were fit with non-linear regression (exponential fit) models; pairwise curve fitting comparisons (extra sum-of-squares F test) are shown in the table at right. D . Kaplan-Meier survival curves of KP mice treated with minoxidil, alone or in combination with α-Pd1 checkpoint therapy. Statistics are not shown because data from the control and α-Pd1-alone groups are from a historical cohort first reported in. 16 PLOD2 inhibition alters fibrillar collagen architecture in UPS Previous reports have shown that fibrillar collagen organization is a critical determinant of CD8 + T cell activation/function in the UPS TME 16 , and that the lysyl hydroxylase activity of Plod2 is critical for maintaining the structure of mature collagen molecules 21 . Therefore, we explored the effects of Plod2 inhibition on collagen fiber organization in the UPS ECM. To this end, we treated mice bearing syngeneic subcutaneous KP tumors with minoxidil or vehicle control and analyzed the architecture of fibrillar collagen molecules in excised tumor sections using multiphoton second-harmonic generation (SHG) imaging. We observed that tumors from minoxidil-treated animals exhibited significantly thinner fibers than those from the control group ( Fig. 3A-B ). Moreover, by measuring the fiber angle relative to the channel direction (x-axis) and plotting the angle frequency against its distribution, we determined that a substantial portion of collagen fibers in control tumors were aligned with each other in a parallel orientation ( Fig. 3C-E ). In contrast, fibers in minoxidil-treated tumors appeared to have a less uniform orientation. Thus, we conclude that Plod2 inhibition remodels fibrillar collagen molecules in the UPS TME, with possible implications for CD8 + T cell function. Download figure Open in new tab Figure 3: Pharmacologic PLOD2 inhibition alters fibrillar collagen architecture in UPS. A . Violin plots depicting CT-FIRE analysis of fibrillar collagen width from tumor sections from mice bearing subcutaneous syngeneic KP tumors treated with minoxidil or control (second harmonic generation [SHG] imaging). Numbers above violin plots indicate means. Thick and thin dotted lines within each plot denote medians and quartiles 1 and 3, respectively. Two-tailed unpaired t-test. B . Representative SHG images of tumors from A . Brightness and contrast have been adjusted for presentation purposes. C . Frequency distribution histograms of fibrillar collagen fiber orientation in tumors from A-B (all microscopy fields combined). Kolmogorov-Smirnov test to compare distributions. D-E . Frequency distribution histograms of fibrillar collagen fiber orientation in individual microscopy fields of control ( D ) and minoxidil-treated ( E ) tumors from C . Alternating light and dark colors in each plot are meant to enable visual separation of individual fields. For A-E , fiber width and orientation were plotted from 6 independent fields across 4 tumors per condition (total n = 24 images/condition). PLOD2 is highly expressed and associated with a poor prognosis in STS To understand the potential clinical utility of PLOD2 inhibitors for UPS, we characterized PLOD2 gene expression patterns in UPS and normal connective tissue specimens. We also included samples from other sarcoma subtypes in this analysis to explore the potential applicability of this approach to a broader swathe of patients with mesenchymal tumors. Using data from multiple human patient cohorts, including the Detwiller et al. dataset 31 and surgical specimens from the Hospital of the University of Pennsylvania (HUP), we observed that PLOD2 gene expression levels were generally upregulated in UPS relative to normal connective tissue samples ( Fig. 4A-B ). Conversely, PLOD2 levels in other sarcoma subtypes such as myxofibrosarcoma (MFS), liposarcoma (LPS), and synovial sarcoma (SS) were more heterogeneous. Consistent with these observations, high levels of PLOD2 expression were associated with significantly reduced disease-free, disease-specific, and overall survival among UPS patients in The Cancer Genome Atlas-Sarcoma (TCGA-SARC) dataset ( Fig. 4C ). Similar, albeit attenuated, relationships were observed among all TCGA-SARC patients ( Fig. 4D ). Thus, modulation of PLOD2 expression and/or activity may improve clinical outcomes in UPS patients, and potentially those with other sarcoma subtypes. Download figure Open in new tab Figure 4: PLOD2 is highly expressed and associated with a poor prognosis in STS. A . PLOD2 gene expression levels in sarcoma and normal connective tissue specimens from the Detwiller et al. data set. 31 MFS: myxofibrosarcoma; LPS: liposarcoma; SS: synovial sarcoma. B . qRT-PCR analysis of PLOD2 expression levels in human sarcoma and normal skeletal muscle tissue specimens (Hospital of the University of Pennsylvania [HUP]). C . Kaplan-Meier survival curves of UPS patients in TCGA-SARC stratified by intratumoral PLOD2 gene expression levels. D . Kaplan-Meier survival curves of all patients in TCGA-SARC stratified by intratumoral PLOD2 gene expression levels. Discussion Over the last 20 years, studies in multiple cancer types have shown that secretion of matrix proteins into the primary tumor milieu impacts tumor progression 35 , 36 . Specifically, collagen fibers can physically associate with cancer cells and promote their migration and invasion 36 . Collagens are also capable of signaling to other cells in the microenvironment including immune cells and platelets 16 , 37 - 39 . In many carcinomas, recruited stromal cells, including activated fibroblasts, secrete ECM proteins 40 . More aggressive epithelial cancer cells, particularly those that have undergone epithelial to mesenchymal transition (EMT), often can secrete ECM as well. 41 However, given the mesenchymal origins of sarcomas, these cells are intrinsically migratory and invasive and secrete large amounts of matrix, promoting metastasis 20 . Critically, matrix protein function in both normal and tumor tissues is influenced primarily by significant PTMs. 19 , 20 , 36 , 42 - 44 These PTMs dramatically alter collagen structure, organization, and signaling to various cell types. In particular, the hypoxia-inducible collagen-modifying enzyme PLOD2 is a collagen lysyl hydroxylase required for collagen production in normal tissues. However, aberrant PLOD2 overexpression in solid tumors such as UPS, as well as bladder, breast, liver, and other carcinomas, promotes cancer cell metastasis and is associated with reduced long-term patient survival 24 , 45 , 46 . Genetic inhibition of Plod2 and treatment with the pan-Plod inhibitor minoxidil dramatically inhibits metastasis in immunodeficient models. 20 Mechanistically, excessive collagen lysyl hydroxylation due to hypoxic induction of PLOD2 results in secretion of immature collagen aggregates, which cancer cells can utilize to facilitate their migration toward blood vessels and entrance (intravasation) into the vasculature 20 . Aberrant Plod2 expression also promotes secretion of lysyl-hydroxylated ColVI into the vasculature, promoting lung endothelial barrier dysfunction and metastasis 21 . However, the effects of UPS cell-derived Plod2 on collagen signaling to immune cells have remained unclear. Recently, several groups have identified mechanisms by which matrix organization and turnover impact immune evasion in the TME 16 , 47 - 49 . These findings led us to query the impact of aberrant PLOD2 expression in primary UPS tumors on the activation status of CD8+ T cells. Here, we report that Plod2 genetic depletion or pan-Plod pharmacologic inhibition in syngeneic UPS allografts abolished primary tumor growth. Importantly, Plod2-dependent tumor growth is unique to immune-competent systems. Specifically, Plod2-deficient UPS xenografts implanted in B and T cell-deficient nude mice grow at the same rate as controls 20 , suggesting that Plod2 suppresses the function of the adaptive immune system in the TME of primary UPS tumors. Consistent with these observations, high PLOD2 expression in human UPS is associated with poor survival. These critical findings suggest that PLOD2 inhibition may augment ICI therapy in UPS patients. Recent clinical trials have demonstrated that the overall response rate of UPS patients to ICI (Pembrolizumab) treatment is only ∼25% 10 , 50 . Development of novel strategies to improve ICI efficacy would be transformative for patients with UPS, and potentially other solid tumors. However, molecular mechanisms underlying ICI response heterogeneity in solid tumors, including UPS, remain poorly understood. Herein, we have demonstrated that sarcoma cell-intrinsic PLOD2 suppresses T cell function using a TnMUC1-CART system. We also tested the effect of pan-PLOD inhibition combined with ICI in both syngeneic and GEMM models of UPS, and found that combination treatment was substantially more effective than either treatment alone. These findings are consistent with the idea that PLOD2 promotes UPS tumor growth by enhancing T cell dysfunction and immune evasion. Our data also implicate PLOD2-mediated alterations in collagen organization as a potential mechanism underlying PLOD2-driven CD8+ T cell inactivation. Therefore, targeting collagen-modifying enzymes such as PLOD2 represents a potential actionable intervention for augmenting immunotherapy responses in UPS patients. In addition to pursuing PLOD2 inhibitors, which are currently in development 51 , there is also a need for an appropriate biomarker to identify appropriate patient cohorts for PLOD2-targeting therapies. Some biomarker possibilities include levels of PLOD2 expression in primary tumor biopsies by IHC, levels of circulating collagens such as the oncogenic PLOD2 substrate COLVI 16 , 21 , or novel approaches to detect excess lysyl hydroxylation of collagen in tissue or blood. Ultimately, the studies described here support the development of matrix-specific and novel immunotherapy strategies for patients with UPS, and potentially other sarcomas and carcinomas. Author contributions Conceptualization: TSKEM, YL, HP Methodology: AMF, HP, YL, EFW Validation: AMF, HP, YL, Formal Analysis: AMF, HP, YL, EFW Investigation: HP, AMF, YL, EFW Data Curation: AMF, YL Provision of resources: JAF, TSKEM Writing-original draft preparation: AMF, HP, YL, TSKEM Writing-review and editing: AMF, HP, YL, TSKEM Visualization: AMF, YL, TSKEM Supervision: JAF, TSKEM Project administration: TSKEM Funding acquisition: TSKEM Acknowledgements We would like to thank James Hayden and Frederick Keeney of the Wistar Institute Imaging Facility for their assistance with multiphoton microscopy and analysis. This work was funded by The University of Pennsylvania Abramson Cancer Center, The Penn Sarcoma Program, Steps to Cure Sarcoma, DoD IDA award RA200237, and NIH/NCI R01CA229688. References 1. ↵ Darvin P , Toor SM , Sasidharan Nair V , Elkord E. Immune checkpoint inhibitors: recent progress and potential biomarkers . Exp Mol Med . 2018 ; 50 ( 12 ): 1 – 11 . Epub 20181213. doi: 10.1038/s12276-018-0191-1 . PubMed PMID: 30546008 ; PMCID: PMC6292890 . OpenUrl CrossRef PubMed 2. ↵ Qin S , Xu L , Yi M , Yu S , Wu K , Luo S. Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4 . Mol Cancer . 2019 ; 18 ( 1 ): 155 . Epub 20191106. doi: 10.1186/s12943-019-1091-2 . PubMed PMID: 31690319 ; PMCID: PMC6833286 . OpenUrl CrossRef PubMed 3. ↵ Sledzinska A , Menger L , Bergerhoff K , Peggs KS , Quezada SA . Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy . Mol Oncol . 2015 ; 9 ( 10 ): 1936 – 65 . Epub 20151026. doi: 10.1016/j.molonc.2015.10.008 . PubMed PMID: 26578451 ; PMCID: PMC5528732 . OpenUrl CrossRef PubMed 4. ↵ Das S , Johnson DB . Immune-related adverse events and anti-tumor efficacy of immune checkpoint inhibitors . J Immunother Cancer . 2019 ; 7 ( 1 ): 306 . Epub 20191115. doi: 10.1186/s40425-019-0805-8 . PubMed PMID: 31730012 ; PMCID: PMC6858629 . OpenUrl Abstract / FREE Full Text 5. ↵ Taylor BS , Barretina J , Maki RG , Antonescu CR , Singer S , Ladanyi M. Advances in sarcoma genomics and new therapeutic targets . Nat Rev Cancer . 2011 ; 11 ( 8 ): 541 – 57 . Epub 2011/07/15. doi: 10.1038/nrc3087nrc3087 [pii]. PubMed PMID: 21753790 ; PMCID: PMC3361898 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Katz D , Palmerini E , Pollack SM . More Than 50 Subtypes of Soft Tissue Sarcoma: Paving the Path for Histology-Driven Treatments . Am Soc Clin Oncol Educ Book . 2018 ; 38 : 925 – 38 . doi: 10.1200/EDBK_205423 . PubMed PMID: 30231352 . OpenUrl CrossRef PubMed 7. ↵ Fletcher CD . The evolving classification of soft tissue tumours - an update based on the new 2013 WHO classification . Histopathology . 2014 ; 64 ( 1 ): 2 – 11 . Epub 20131025. doi: 10.1111/his.12267 . PubMed PMID: 24164390 . OpenUrl CrossRef PubMed 8. ↵ Potter JW , Jones KB , Barrott JJ . Sarcoma-The standard-bearer in cancer discovery . Crit Rev Oncol Hematol . 2018 ; 126 : 1 – 5 . Epub 20180329. doi: 10.1016/j.critrevonc.2018.03.007 . PubMed PMID: 29759550 ; PMCID: PMC5961738 . OpenUrl CrossRef PubMed 9. ↵ Cancer Genome Atlas Research Network . Electronic address edsc, Cancer Genome Atlas Research N . Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas. Cell . 2017 ; 171 ( 4 ): 950 – 65 e28. doi: 10.1016/j.cell.2017.10.014 . PubMed PMID: 29100075 ; PMCID: PMC5693358 . OpenUrl CrossRef PubMed 10. ↵ Tawbi HA , Burgess M , Bolejack V , Van Tine BA , Schuetze SM , Hu J , D’Angelo S , Attia S , Riedel RF , Priebat DA , Movva S , Davis LE , Okuno SH , Reed DR , Crowley J , Butterfield LH , Salazar R , Rodriguez-Canales J , Lazar AJ , Wistuba , II , Baker LH , Maki RG , Reinke D , Patel S. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial . Lancet Oncol . 2017 ; 18 ( 11 ): 1493 – 501 . Epub 20171004. doi: 10.1016/S1470-2045(17)30624-1 . PubMed PMID: 28988646 ; PMCID: PMC7939029 . OpenUrl CrossRef PubMed 11. ↵ Widemann BC , Italiano A. Biology and Management of Undifferentiated Pleomorphic Sarcoma, Myxofibrosarcoma, and Malignant Peripheral Nerve Sheath Tumors: State of the Art and Perspectives . J Clin Oncol . 2018 ; 36 ( 2 ): 160 – 7 . Epub 20171208. doi: 10.1200/JCO.2017.75.3467 . PubMed PMID: 29220302 ; PMCID: PMC5759316 . OpenUrl CrossRef PubMed 12. Camargo FD , Gokhale S , Johnnidis JB , Fu D , Bell GW , Jaenisch R , Brummelkamp TR . YAP1 increases organ size and expands undifferentiated progenitor cells . Curr Biol . 2007 ; 17 ( 23 ): 2054 – 60 . doi: 10.1016/j.cub.2007.10.039 . PubMed PMID: 17980593 . OpenUrl CrossRef PubMed Web of Science 13. Pan D. The hippo signaling pathway in development and cancer . Dev Cell . 2010 ; 19 ( 4 ): 491 – 505 . Epub 2010/10/19. doi: 10.1016/j.devcel.2010.09.011S1534-5807(10)00429-6 [pii]. PubMed PMID: 20951342 ; PMCID: PMC3124840 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Zhao B , Li L , Lei Q , Guan KL . The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version . Genes Dev . 2010 ; 24 ( 9 ): 862 – 74 . doi: 10.1101/gad.1909210 . PubMed PMID: 20439427 ; PMCID: PMC2861185 . OpenUrl Abstract / FREE Full Text 15. ↵ Ricard-Blum S. The collagen family . Cold Spring Harb Perspect Biol . 2011 ; 3 ( 1 ): a004978 . Epub 20110101. doi: 10.1101/cshperspect.a004978 . PubMed PMID: 21421911 ; PMCID: PMC3003457 . OpenUrl Abstract / FREE Full Text 16. ↵ Fuller AM , Pruitt HC , Liu Y , Irizarry-Negron VM , Pan H , Song H , DeVine A , Katti RS , Devalaraja S , Ciotti GE , Gonzalez MV , Williams EF , Murazzi I , Ntekoumes D , Skuli N , Hakonarson H , Zabransky DJ , Trevino JG , Weeraratna A , Weber K , Haldar M , Fraietta JA , Gerecht S , Eisinger-Mathason TSK. Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment . J Clin Invest . 2024 ; 134 ( 11 ). Epub 20240423. doi: 10.1172/JCI167826 . PubMed PMID: 38652549 ; PMCID: PMC11142734 . OpenUrl CrossRef PubMed 17. ↵ Pruitt HC , Guan Y , Liu H , Carey AE , Brennen WN , Lu J , Joshu C , Weeraratna A , Lotan TL , Karin Eisinger-Mathason TS , Gerecht S. Collagen VI deposition mediates stromal T cell trapping through inhibition of T cell motility in the prostate tumor microenvironment . Matrix Biol . 2023 ; 121 : 90 – 104 . Epub 20230616. doi: 10.1016/j.matbio.2023.06.002 . PubMed PMID: 37331435 . OpenUrl CrossRef PubMed 18. ↵ Schwarze U , Cundy T , Pyott SM , Christiansen HE , Hegde MR , Bank RA , Pals G , Ankala A , Conneely K , Seaver L , Yandow SM , Raney E , Babovic-Vuksanovic D , Stoler J , Ben-Neriah Z , Segel R , Lieberman S , Siderius L , Al-Aqeel A , Hannibal M , Hudgins L , McPherson E , Clemens M , Sussman MD , Steiner RD , Mahan J , Smith R , Anyane-Yeboa K , Wynn J , Chong K , Uster T , Aftimos S , Sutton VR , Davis EC , Kim LS , Weis MA , Eyre D , Byers PH . Mutations in FKBP10, which result in Bruck syndrome and recessive forms of osteogenesis imperfecta, inhibit the hydroxylation of telopeptide lysines in bone collagen . Hum Mol Genet . 2013 ; 22 ( 1 ): 1 – 17 . Epub 2012/09/06. doi: 10.1093/hmg/dds371dds371 [pii]. PubMed PMID: 22949511 ; PMCID: PMC3606010 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Bank RA , Robins SP , Wijmenga C , Breslau-Siderius LJ , Bardoel AF , van der Sluijs HA , Pruijs HE , TeKoppele JM . Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome Proc Natl Acad Sci U S A . 1999 ; 96 ( 3 ): 1054 – 8 . doi: 10.1073/pnas.96.3.1054 . PubMed PMID: 9927692 ; PMCID: PMC15349 . OpenUrl Abstract / FREE Full Text 20. ↵ Eisinger-Mathason TS , Zhang M , Qiu Q , Skuli N , Nakazawa MS , Karakasheva T , Mucaj V , Shay JE , Stangenberg L , Sadri N , Pure E , Yoon SS , Kirsch DG , Simon MC . Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis . Cancer Discov . 2013 ; 3 ( 10 ): 1190 – 205 . doi: 10.1158/2159-8290.CD-13-0118 . PubMed PMID: 23906982 ; PMCID: 3822914 . OpenUrl Abstract / FREE Full Text 21. ↵ Liu Y , Murazzi I , Fuller AM , Pan H , Irizarry-Negron VM , Devine A , Katti R , Skuli N , Ciotti GE , Pak K , Pack MA , Simon MC , Weber K , Cooper K , Eisinger-Mathason TSK . Sarcoma Cells Secrete Hypoxia-Modified Collagen VI to Weaken the Lung Endothelial Barrier and Promote Metastasis . Cancer Res . 2024 ; 84 ( 7 ): 977 – 93 . doi: 10.1158/0008-5472.CAN-23-0910 . PubMed PMID: 38335278 ; PMCID: PMC10984776 . OpenUrl CrossRef PubMed 22. Lewis DM , Pruitt H , Jain N , Ciccaglione M , McCaffery JM , Xia Z , Weber K , Eisinger-Mathason TSK , Gerecht S. A Feedback Loop between Hypoxia and Matrix Stress Relaxation Increases Oxygen-Axis Migration and Metastasis in Sarcoma . Cancer Res . 2019 ; 79 ( 8 ): 1981 – 95 . Epub 2019/02/20. doi: 10.1158/0008-5472.CAN-18-1984 . PubMed PMID: 30777851 ; PMCID: PMC6727644 . OpenUrl Abstract / FREE Full Text 23. Li K , Niu Y , Li K , Zhong C , Qiu Z , Yuan Y , Shi Y , Lin Z , Huang Z , Zuo D , Yuan Y , Li B. Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma . J Clin Transl Hepatol . 2023 ; 11 ( 5 ): 1094 – 105 . Epub 20230421. doi: 10.14218/JCTH.2022.00401 . PubMed PMID: 37577214 ; PMCID: PMC10412693 . OpenUrl CrossRef PubMed 24. ↵ Miyamoto K , Seki N , Matsushita R , Yonemori M , Yoshino H , Nakagawa M , Enokida H. Tumour-suppressive miRNA-26a-5p and miR-26b-5p inhibit cell aggressiveness by regulating PLOD2 in bladder cancer . Br J Cancer . 2016 ; 115 ( 3 ): 354 – 63 . Epub 20160616. doi: 10.1038/bjc.2016.179 . PubMed PMID: 27310702 ; PMCID: PMC4973152 . OpenUrl CrossRef PubMed 25. Kurozumi A , Kato M , Goto Y , Matsushita R , Nishikawa R , Okato A , Fukumoto I , Ichikawa T , Seki N. Regulation of the collagen cross-linking enzymes LOXL2 and PLOD2 by tumor-suppressive microRNA-26a/b in renal cell carcinoma . Int J Oncol . 2016 ; 48 ( 5 ): 1837 – 46 . Epub 20160315. doi: 10.3892/ijo.2016.3440 . PubMed PMID: 26983694 ; PMCID: PMC4809659 . OpenUrl CrossRef PubMed 26. Blanco MA , LeRoy G , Khan Z , Aleckovic M , Zee BM , Garcia BA , Kang Y. Global secretome analysis identifies novel mediators of bone metastasis . Cell Res . 2012 ; 22 ( 9 ): 1339 – 55 . Epub 20120612. doi: 10.1038/cr.2012.89 . PubMed PMID: 22688892 ; PMCID: PMC3434351 . OpenUrl CrossRef PubMed 27. Ueki Y , Saito K , Iioka H , Sakamoto I , Kanda Y , Sakaguchi M , Horii A , Kondo E. PLOD2 Is Essential to Functional Activation of Integrin beta1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas . iScience . 2020 ; 23 ( 2 ): 100850 . Epub 20200118. doi: 10.1016/j.isci.2020.100850 . PubMed PMID: 32058962 ; PMCID: PMC6997870 . OpenUrl CrossRef PubMed 28. ↵ Wan J , Qin J , Cao Q , Hu P , Zhong C , Tu C. Hypoxia-induced PLOD2 regulates invasion and epithelial-mesenchymal transition in endometrial carcinoma cells . Genes Genomics . 2020 ; 42 ( 3 ): 317 – 24 . Epub 20191223. doi: 10.1007/s13258-019-00901-y . PubMed PMID: 31872384 . OpenUrl CrossRef PubMed 29. ↵ Kirsch DG , Dinulescu DM , Miller JB , Grimm J , Santiago PM , Young NP , Nielsen GP , Quade BJ , Chaber CJ , Schultz CP , Takeuchi O , Bronson RT , Crowley D , Korsmeyer SJ , Yoon SS , Hornicek FJ , Weissleder R , Jacks T. A spatially and temporally restricted mouse model of soft tissue sarcoma . Nat Med . 2007 ; 13 ( 8 ): 992 – 7 . Epub 2007/08/07. doi: nm1602 [pii] 10.1038/nm1602 . PubMed PMID: 17676052 . OpenUrl CrossRef PubMed Web of Science 30. ↵ Bredfeldt JS , Liu Y , Pehlke CA , Conklin MW , Szulczewski JM , Inman DR , Keely PJ , Nowak RD , Mackie TR , Eliceiri KW . Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer . J Biomed Opt . 2014 ; 19 ( 1 ): 16007 . doi: 10.1117/1.JBO.19.1.016007 . PubMed PMID: 24407500 ; PMCID: PMC3886580 . OpenUrl CrossRef PubMed 31. ↵ Detwiller KY , Fernando NT , Segal NH , Ryeom SW , D’Amore PA , Yoon SS . Analysis of hypoxia-related gene expression in sarcomas and effect of hypoxia on RNA interference of vascular endothelial cell growth factor A . Cancer Res . 2005 ; 65 ( 13 ): 5881 – 9 . Epub 2005/07/05. doi: 65/13/5881 [pii] 10.1158/0008-5472.CAN-04-4078 . PubMed PMID: 15994966 . OpenUrl Abstract / FREE Full Text 32. ↵ Zuurmond AM , van der Slot-Verhoeven AJ , van Dura EA , De Groot J , Bank RA . Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis . Matrix Biol . 2005 ; 24 ( 4 ): 261 – 70 . doi: 10.1016/j.matbio.2005.04.002 . PubMed PMID: 15908192 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Waldman AD , Fritz JM , Lenardo MJ . A guide to cancer immunotherapy: from T cell basic science to clinical practice . Nat Rev Immunol . 2020 ; 20 ( 11 ): 651 – 68 . Epub 20200520. doi: 10.1038/s41577-020-0306-5 . PubMed PMID: 32433532 ; PMCID: PMC7238960 . OpenUrl CrossRef PubMed 34. ↵ Posey AD , Jr . ., Schwab RD , Boesteanu AC , Steentoft C , Mandel U , Engels B , Stone JD , Madsen TD , Schreiber K , Haines KM , Cogdill AP , Chen TJ , Song D , Scholler J , Kranz DM , Feldman MD , Young R , Keith B , Schreiber H , Clausen H , Johnson LA , June CH . Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma . Immunity . 2016 ; 44 ( 6 ): 1444 – 54 . doi: 10.1016/j.immuni.2016.05.014 . PubMed PMID: 27332733 ; PMCID: PMC5358667 . OpenUrl CrossRef PubMed 35. ↵ Fang M , Yuan J , Peng C , Li Y. Collagen as a double-edged sword in tumor progression . Tumour Biol . 2014 ; 35 ( 4 ): 2871 – 82 . doi: 10.1007/s13277-013-1511-7 . PubMed PMID: 24338768 ; PMCID: 3980040 . OpenUrl CrossRef PubMed 36. ↵ Han W , Chen S , Yuan W , Fan Q , Tian J , Wang X , Chen L , Zhang X , Wei W , Liu R , Qu J , Jiao Y , Austin RH , Liu L. Oriented collagen fibers direct tumor cell intravasation . Proc Natl Acad Sci U S A . 2016 ; 113 ( 40 ): 11208 – 13 . doi: 10.1073/pnas.1610347113 . PubMed PMID: 27663743 ; PMCID: 5056065 . OpenUrl Abstract / FREE Full Text 37. ↵ Dumont B , Lasne D , Rothschild C , Bouabdelli M , Ollivier V , Oudin C , Ajzenberg N , Grandchamp B , Jandrot-Perrus M. Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations . Blood . 2009 ; 114 ( 9 ): 1900 – 3 . doi: 10.1182/blood-2009-03-213504 . PubMed PMID: 19549989 . OpenUrl Abstract / FREE Full Text 38. Smethurst PA , Onley DJ , Jarvis GE , O’Connor MN , Knight CG , Herr AB , Ouwehand WH , Farndale RW . Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets . J Biol Chem . 2007 ; 282 ( 2 ): 1296 – 304 . Epub 2006/11/07. doi: M606479200 [pii] 10.1074/jbc.M606479200 . PubMed PMID: 17085439 . OpenUrl Abstract / FREE Full Text 39. ↵ Farndale RW . Collagen-induced platelet activation . Blood Cells Mol Dis . 2006 ; 36 ( 2 ): 162 – 5 . Epub 2006/02/09. doi: S1079-9796(06)00017-9 [pii] 10.1016/j.bcmd.2005.12.016 . PubMed PMID: 16464621 . OpenUrl CrossRef PubMed 40. ↵ Kalluri R , Zeisberg M. Fibroblasts in cancer . Nat Rev Cancer . 2006 ; 6 ( 5 ): 392 – 401 . Epub 2006/03/31. doi: nrc1877 [pii] 10.1038/nrc1877 . PubMed PMID: 16572188 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Neri S , Miyashita T , Hashimoto H , Suda Y , Ishibashi M , Kii H , Watanabe H , Kuwata T , Tsuboi M , Goto K , Menju T , Sonobe M , Date H , Ochiai A , Ishii G. Fibroblast-led cancer cell invasion is activated by epithelial-mesenchymal transition through platelet-derived growth factor BB secretion of lung adenocarcinoma . Cancer Lett . 2017 ; 395 : 20 – 30 . doi: 10.1016/j.canlet.2017.02.026 . PubMed PMID: 28286261 . OpenUrl CrossRef PubMed 42. ↵ Akiri G , Sabo E , Dafni H , Vadasz Z , Kartvelishvily Y , Gan N , Kessler O , Cohen T , Resnick M , Neeman M , Neufeld G. Lysyl oxidase-related protein-1 promotes tumor fibrosis and tumor progression in vivo . Cancer Res . 2003 ; 63 ( 7 ): 1657 – 66 . Epub 2003/04/03. PubMed PMID: 12670920 . OpenUrl Abstract / FREE Full Text 43. Gilkes DM , Chaturvedi P , Bajpai S , Wong CC , Wei H , Pitcairn S , Hubbi ME , Wirtz D , Semenza GL . Collagen Prolyl Hydroxylases Are Essential for Breast Cancer Metastasis . Cancer Res . 2013 . Epub 2013/03/30. doi: 0008-5472.CAN-12-3963 [pii] 10.1158/0008-5472.CAN-12-3963 . PubMed PMID: 23539444 . OpenUrl Abstract / FREE Full Text 44. ↵ Hofbauer KH , Gess B , Lohaus C , Meyer HE , Katschinski D , Kurtz A. Oxygen tension regulates the expression of a group of procollagen hydroxylases . Eur J Biochem . 2003 ; 270 ( 22 ): 4515 – 22 . Epub 2003/11/19. doi: 3846[pii]. PubMed PMID: 14622280 . OpenUrl CrossRef PubMed Web of Science 45. ↵ Gilkes DM , Bajpai S , Wong CC , Chaturvedi P , Hubbi ME , Wirtz D , Semenza GL . Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis . Mol Cancer Res . 2013 ; 11 ( 5 ): 456 – 66 . Epub 2013/02/05. doi: 10.1158/1541-7786 . MCR-12-06291541-7786.MCR-12-0629 [pii]. PubMed PMID: 23378577 ; PMCID: PMC3656974 . OpenUrl Abstract / FREE Full Text 46. ↵ Noda T , Yamamoto H , Takemasa I , Yamada D , Uemura M , Wada H , Kobayashi S , Marubashi S , Eguchi H , Tanemura M , Umeshita K , Doki Y , Mori M , Nagano H. PLOD2 induced under hypoxia is a novel prognostic factor for hepatocellular carcinoma after curative resection . Liver Int . 2012 ; 32 ( 1 ): 110 – 8 . Epub 2011/11/22. doi: 10.1111/j.1478-3231.2011.02619.x . PubMed PMID: 22098155 . OpenUrl CrossRef PubMed 47. ↵ Tharp KM , Kersten K , Maller O , Timblin GA , Stashko C , Canale FP , Menjivar RE , Hayward MK , Berestjuk I , Ten Hoeve J , Samad B , Ironside AJ , di Magliano MP , Muir A , Geiger R , Combes AJ , Weaver VM . Tumor-associated macrophages restrict CD8(+) T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment . Nat Cancer . 2024 . Epub 20240603. doi: 10.1038/s43018-024-00775-4 . PubMed PMID: 38831058 . OpenUrl CrossRef PubMed 48. LaRue MM , Parker S , Puccini J , Cammer M , Kimmelman AC , Bar-Sagi D. Metabolic reprogramming of tumor-associated macrophages by collagen turnover promotes fibrosis in pancreatic cancer . Proc Natl Acad Sci U S A . 2022 ; 119 ( 16 ): e2119168119 . Epub 20220411. doi: 10.1073/pnas.2119168119 . PubMed PMID: 35412885 ; PMCID: PMC9169723 . OpenUrl CrossRef PubMed 49. ↵ Vijver SV , Singh A , Mommers-Elshof E , Meeldijk J , Copeland R , Boon L , Langermann S , Flies D , Meyaard L , Ramos MIP . Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1 . Front Immunol . 2021 ; 12 : 733561 . Epub 20211007. doi: 10.3389/fimmu.2021.733561 . PubMed PMID: 34691040 ; PMCID: PMC8529287 . OpenUrl CrossRef PubMed 50. ↵ Keung EZ , Lazar AJ , Torres KE , Wang WL , Cormier JN , Ashleigh Guadagnolo B , Bishop AJ , Lin H , Hunt KK , Bird J , Lewis VO , Patel SR , Wargo JA , Somaiah N , Roland CL . Phase II study of neoadjuvant checkpoint blockade in patients with surgically resectable undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma . BMC Cancer . 2018 ; 18 ( 1 ): 913 . Epub 20180924. doi: 10.1186/s12885-018-4829-0 . PubMed PMID: 30249211 ; PMCID: PMC6154892 . OpenUrl CrossRef PubMed 51. ↵ Maghsoud Y , Vazquez-Montelongo EA , Yang X , Liu C , Jing Z , Lee J , Harger M , Smith AK , Espinoza M , Guo HF , Kurie JM , Dalby KN , Ren P , Cisneros GA . Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition . J Chem Inf Model . 2023 ; 63 ( 3 ): 986 – 1001 . Epub 20230130. doi: 10.1021/acs.jcim.2c01448 . PubMed PMID: 36779232 ; PMCID: PMC10233724 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 02, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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