{"paper_id":"85e4fb3d-36ab-4a78-ac9e-ea69ee0bf794","body_text":"RESEARCH Open Access\n© The Author(s) 2025. Open Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 \nInternational License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you \ngive appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the \nlicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. 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BMC Cancer         (2025) 25:1674 \nhttps://doi.org/10.1186/s12885-025-15108-6\nBMC Cancer\n*Correspondence:\nGuiying Nie\nguiying.nie@rmit.edu.au\n1Implantation and Pregnancy Research Laboratory, School of Health and \nBiomedical Sciences, RMIT University, Bundoora, VIC 3083, Australia\n2Ageing and Immunotherapies Research Group, School of Health and \nBiomedical Sciences, RMIT University, Bundoora, VIC 3083, Australia\n3Department of Biochemistry and Molecular Biology, Monash \nBiomedicine Discovery Institute, Monash University, Clayton, VIC  \n3800, Australia\n4Hudson Institute of Medical Research, Clayton, VIC 3168, Australia\n5Department of Molecular and Translational Sciences, Monash University, \nClayton, VIC 3168, Australia\nAbstract\nBackground High grade serous carcinoma (HGSC) is the most common and lethal subtype of ovarian cancer, yet \nits prognosis has remained unchanged in the past 3 decades. HGSC is known to have evolved immune evasion \nstrategies to promote survival, but these mechanisms are not well understood. Podocalyxin (PODXL), a CD34-related \nsialomucin, is often expressed in HGSC patients with poor prognosis. We have recently reported that PODXL promotes \nthe formation of compact and chemoresistant HGSC spheroids to boost their survival.\nMethods In this current study, we investigated whether PODXL may also influence HGSC spheroid susceptibility \nto NK cell infiltration and cytotoxicity. We co-cultured HGSC spheroids with primary human NK cells isolated from \nperipheral blood mononuclear cells (PBMCs) and examined the impact on these spheroids following 24, 48 and 72 h \nof co-culture. We first used a cell line model of HGSC spheroids employing Kuramochi cells, which express the highest \nlevel of PODXL among known HGSC cell lines. To study the impact of PODXL levels, we compared spheroids of control \nand PODXL knockout (PODXL-KO) cells that we have previously engineered. We then validated the data in primary \ncancer spheroids derived from ascites of HGSC patients that express high and low levels of PODXL.\nResults In both the cell line and primary HGSC spheroid models, co-culture of spheroids expressing lower levels of \nPODXL resulted in more NK cell infiltration and cytotoxicity, while spheroids expressing higher levels of PODXL were \nresistant to destruction and showed more proliferation.\nConclusions Collectively, these data suggest that PODXL may play an important role in aiding immune evasion in \nHGSC, at least partly by conferring resistance to NK cell infiltration and the related cytotoxicity.\nKeywords HGSC, Spheroids, Podocalyxin, PODXL, Immunity, NK cells, Infiltration\nPodocalyxin protects high grade serous \novarian cancer spheroids from NK cell \ninfiltration and spheroid destruction\nNgoc Le Tran1, Yao Wang1, Kylie M. Quinn2,3, Maree Bilandzic4,5, Andrew Stephens4,5 and Guiying Nie1*\n\nPage 2 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nIntroduction\nOvarian cancer (OC) remains the most lethal gynaeco -\nlogical malignancy worldwide with a 5-year survival rate \nbelow 50% [ 1]. High grade serous carcinoma (HGSC), \naccounting for over 90% of OC cases, is an aggressive \nand heterogenous disease that is characterised by high \nmortality, late diagnosis and propensity for recurrence \ndue to chemoresistance [ 2, 3]. The first line of treatment \nincludes a combination of debulking surgery, and a plati -\nnum and/taxane-based chemotherapy regimen [4]. While \naround 70% of patients respond to the treatment, the \nmajority of these subsequently unfortunately experience \ncancer recurrence due to a lack of effective maintenance \ntherapies and the drug-resistant nature of the tumors \n[5]. Therefore, there is an urgent need to develop new \ntherapeutics to improve the prognosis of OC, especially \nHGSC.\nHGSC spreads predominantly in the peritoneal cav -\nity whereby exfoliated tumor cells travel via ascites fluid \nto secondary sites [ 6]. Ascites, present in more than one \nthird of OC patients, acts as a reservoir for various cel -\nlular components including tumor cells, cancer associ -\nated fibroblasts, mesothelial cells, and immune cells [ 7]. \nSeveral studies have observed that immune cell infiltra -\ntion into the tumor microenvironment, where malignant \nascites accumulates, is correlated with good prognosis of \nOC [8, 9]. For instance, the presence of CD3 + T cells in \nthe tumor is correlated with increased survival in HGSC \npatients [10]. Studies also indicate the importance of nat-\nural killer (NK) cells, although the precise mechanism of \nhow NK cells can enhance patient prognosis is still to be \nevaluated [11– 13].\nNK cells, effector lymphocytes of the innate system, \nplay a pivotal role in inducing cytolytic activity against \naberrant cells caused by viral infections and cancer [ 14]. \nThe ability of NK cells to exhibit antitumor effects with -\nout priming or prior activation is advantageous com -\npared to other immune cells, which are restricted by \npatient-specific major histocompatibility complex mol -\necules to elicit an immune response [ 15, 16]. Therefore, \nNK cell-based adoptive cellular immunotherapy has \nbecome increasingly popular [ 17]. NK cell activation is \ntightly controlled by a range of activating and inhibitory \nreceptors that interact with respective ligands expressed \non target cells [ 18]. Once activated, NK cells can exert \ncytolytic effects through various effector mechanisms \nsuch as antibody dependent cellular cytotoxicity, cyto -\ntoxic granules, and secretion of inflammatory cytokine or \nchemokines [19– 21]. These diverse functions of NK cells \nin immune surveillance may allow a potentially impor -\ntant therapeutic strategy for cancer immunotherapy. \nIndeed, the presence of NK cells within the immune rep -\nertoire of ascites is reported to be positively correlated to \nbetter outcomes in OC patients [15]. However, the killing \ncapacity of NK cells can be mitigated by several immune \nevasion strategies of the cancer itself, including the estab-\nlishment of an immunosuppressive milieu, which pro -\nmotes cancer cell survival, metastasis and invasion [22].\nPodocalyxin (PODXL) is a sialomucin normally \nexpressed on the surface of various cells including kid -\nney glomeruli, epithelial and endothelial cells, meso -\nthelium, and hematopoietic progenitor cells [ 23]. High \nPODXL expression has been associated with aggressive \ntumor phenotypes and poor prognosis in several cancers \nincluding breast, pancreatic and ovarian cancer [ 23– 27]. \nIn OCs, PODXL is more likely to be expressed in HGSC \n(87%) compared to other subtypes, and its surface \nexpression is more associated with late stage HGSC and a \nsignificant decrease in disease-free survival [ 25, 28].\nFunctionally, we have reported that in HGSC, PODXL \npromotes the formation of compact and chemoresistant \ncancer spheroids [ 24]. In both HGSC-derived cell lines \nand ascites-derived primary HGSC cells, cancer spher -\noids with higher PODXL expression are more compact \nand less fragile to fragmentation than those of lower \nPODXL levels [ 24]; furthermore, the former were more \nresilient to chemotherapy drugs as they showed higher \ncell proliferation following treatment [ 24]. These results \nsuggest that PODXL increases HGSC spheroid survival \n[24].\nIt is still unknown how PODXL bestows HGSC spher -\noids with such an advantage, but we believe it is inter -\nrelated to the fundamental role of PODXL in promoting \nepithelial barrier functions, which has been recently \nrevealed in endometrial epithelial cells where PODXL \npromotes an impermeable epithelial barrier [ 29]. We \ntherefore hypothesized that PODXL may play a role in \nprotecting cancer spheroids from the surroundings by \nhindering penetration/action of drugs and immune cells \nsuch as NK cells.\nIn this study we aimed to investigate whether PODXL \nlevels in HGSC spheroids influence NK cell infiltration \nand spheroid destruction. We co-cultured HGSC spher -\noids with primary human NK cells isolated from periph -\neral blood mononuclear cells (PBMCs) and examined \ntheir infiltration into the spheroid and related cytotox -\nicity. We first used a cell line model of HGSC spheroids \nemploying Kuramochi cells, which express the high -\nest level of PODXL among known HGSC cell lines [ 24]. \nTo examine the importance of PODXL, we compared \nspheroids of control and PODXL knockout (PODXL-\nKO) Kuramochi cells that we have previously created \nusing CRISPR/Cas9 gene editing [ 24]. We then validated \nthe data in primary cancer spheroids derived from asci -\ntes of HGSC patients that express high and low levels of \nPODXL. Collectively, our results suggest that PODXL \nprotects HGSC spheroids from infiltration and cytotoxic-\nity effects of NK cells.\n\nPage 3 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nMethods\nCulture of Kuramochi and primary HGSC cancer cells\nKuramochi cell line was purchased from CellBank Aus -\ntralia (NSW, Australia) and cultured in RPMI 1640 \nMedium + GlutaMAX supplement (Thermo Fisher Sci -\nentific, MA, USA, #61870036). Control (transfected with \nempty vectors) and PODXL knockout (KO) Kuramochi \ncells were generated as previously described [ 24], and \nmaintained with 1 µg/ml puromycin (Sigma-Aldrich, \n#P8833) that was added into the culture media. Asci -\ntes-derived primary HGSC cells were isolated as previ -\nously described [ 24], and maintained in a 1:1 ratio of \nMedium 199 (Thermo Fisher Scientific, #11150-059) \nand MCDB131 (Thermo Fisher Scientific, #10372-019). \nPrimary cells were screened for CA125 to confirm OC \ncancer origin and were used between passages 4–7. All \nmedia were supplemented with 10% (for Kuramochi \ncells) or 15% (for primary cells) fetal bovine serum (FBS, \nThermo Fisher Scientific) and 1% antibiotic–antimycotic \n(Thermo Fisher Scientific, #15240062); all cells were cul -\ntured at 37 °C under 5% CO2.\nIsolation of primary human NK cells and evaluation by flow \ncytometry\nNK cells were isolated from PBMCs derived from whole \nblood of 4 healthy female volunteer donors (aged 21–28), \nwhich was provided by the Australian Red Cross with \nethics approval by RMIT College of Human Ethics Advi -\nsory Network (#28056). All work was conducted accord -\ning to the Declaration of Helsinki Principles and the \nAustralian National Health and Medical Research Coun -\ncil (NHMRC) Code of Practice. Signed informed consent \nwas obtained from all donors before the study.\nPBMCs were isolated from fresh blood by a density \ngradient centrifugation using Lymphoprep (StemCell \nTechnologies, BC, Canada, #07801) whereby 10 ml of \nLymphoprep was added to a 50 ml tube then overlayed \nwith 25 ml of diluted blood (diluted 1:1 in incomplete \n(i) RPMI). The tube was then centrifuged at 800 xg for \n20 min at 23 °C with brake off. The interface containing \nmononuclear cells was transferred into a fresh 50 ml tube \ncontaining iRPMI, centrifuged at 450 xg for 4 min at 23 \n°C with brake on, washed once, frozen in freezing media \n(10% DMSO in FBS) and stored at ≤−150 °C until further \nuse.\nAt the time of NK cell use, cryopreserved PBMCs were \nthawed and NK cells were isolated using an NK Cell Iso -\nlation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, \n#130092657), LS Column (Miltenyi Biotec, #130122729,) \nand QuadropMACS™ Separator (Miltenyi Biotec, \n#130090976) according to the manufacturers’ proto -\ncol. The enrichment of NK cells was evaluated by stain -\ning for viability with Live/dead NearIR (Thermo Fisher \nScientific, #L34975) followed by anti-CD3 fluorescein \nisothiocyanate (FITC) (Miltenyi Biotec, #130113690) \nand anti-CD56 phycoerythrin (PE) (Miltenyi Biotec, \n#130113874) and analysis on an LSR Fortessa X-20 flow \ncytometer (BD Biosciences, USA). NK cells were defined \nas live cells that were CD3- and CD56+ (Supplementary \nFig. 1) and on average 93% of the enriched cells were con-\nfirmed to be NK cells.\nIsolated NK cells were activated by culturing for 72 h \nat 37 °C under 5% CO 2 in RPMI 1640 Medium + Gluta-\nMAX supplement (Thermo Fisher Scientific, MA, USA, \n#61870036), supplemented with 10% FBS, 1% penicillin-\nstreptomycin, and 10ng/ml IL-15 (Stem cells, #78031.1). \nThey were stained with carboxyfluoroscein succinimidyl \nester (CFSE, Thermo Fisher Scientific, #C34570) for 10 \nmin (diluted 1:1000 in PBS) as per manufacturer’s proto-\ncol before being co-cultured with cancer spheroids.\nCo-culture of cancer spheroids with NK cells\nKuramochi and primary HGSC cancer spheroids were \nformed in Costar ultra-low attachment round bottom \n96 well plates (Merck, Darmstadt, Germany, #CLS7007-\n24EA) by culturing 2,500 cells per well in complete \nmedium for 3 days as previously reported [ 24]. The \nresulting spheroids were then mixed with fluorescently \nlabelled NK cells that were prepared as described above \n(at a ratio of 3:1), and co-cultured for 24, 48 and 72 h \nrespectively. NK cell infiltration into spheroids and can -\ncer cell cytotoxicity were assessed as described below.\nAnalysis of NK cell infiltration and the impact on the \nspheroid size and cell number\nAt each time point of assessment, brightfield images of \nthe co-cultured spheroids were taken using the Nikon \neclipse TS100 microscope equipped with a Nikon DS-Fi1 \ncamera (Tokyo, Japan); thereafter confocal images of \nco-cultured spheroids were taken with an A1R confo -\ncal microscope (Nikon, Japan) to identify the fluores -\ncently labelled NK cells. The co-cultured spheroids were \nthen washed with PBS using a 10 µl pipette tip until all \nattached NK cells on the outside of the spheroids are \ncompletely removed, after which brightfield images of the \nwashed spheroids were taken.\nTo analyse NK cell penetration into the spheroid, total \nNK cell fluorescence intensity within the co-cultured, \nunwashed spheroids was quantified (as washing may \nlead to loss of some NK cells). To do this, the outline of \neach spheroid was determined on the washed spheroid \nthen overlayed onto the confocal image of the unwashed \nspheroid, the total NK cell fluorescence intensity within \nthe spheroid were then determined using the ImageJ \nsoftware version 1.53c (NIH, USA). The final data were \nexpressed as total fluorescence after subtracting back -\nground fluorescence. The experiment was repeated 4 \ntimes with NK cells of 4 different donors ( n = 4). For \n\nPage 4 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \neach experiment with NK cells of one donor, 3 individual \nspheroids were quantified and their average was used as \nthe data. This applies to all other experiments concerning \nquantification following co-culture with NK cells.\nThe volume of spheroids following the PBS wash was \nalso determined at each time point. To do this, the diam -\neter of each spheroid was measured using the average \nlength value of 4 different angles using the “straight line” \nfunction of the ImageJ software, and the spheroid volume \nwas calculated using the formula of 4/3πr 3. The number \nand viability of live cells contained within the washed \nspheroids were also analysed. To do this, the washed \nspheroids were trypsinized and dissociated into single \ncells, they were then mixed with trypan blue (Thermo \nFisher Scientific) and analysed with a Countess 3 auto -\nmated cell counter (Thermo Fisher Scientific).\nAnalysis of NK cell-induced cytotoxicity within spheroids\nSpheroids were formed and co-cultured with fluores -\ncently labelled NK cells for 24, 48 and 72 h respectively \nas described above, they were then incubated for 1 h with \nCellEvent Caspase-3/7 red detection reagent (Thermo \nFisher Scientific, #C10430) (diluted 1:100 in complete \nmedia); after which the spheroids were first imaged using \nan A1R confocal microscope (Nikon, Japan), then disso -\nciated into single cells by gentle pipetting and assessed \nfor fluorescence at ~ 502/530 nm (excitation/emission) \non a CLARIOstar® Plus plate reader (BMG LABTECH, \nOrtenberg, Baden-Württemberg, Germany). As primary \nHGSC spheroids were too small and compact to disso -\nciate by manual pipetting, primary spheroids were mea -\nsured on the plate reader without dissociation.\nAnalysis of Ki-67 positive cancer cells within the co-cultured \nspheroids\nTo assess cancer cell proliferation within spheroids fol -\nlowing co-culturing with NK cells, spheroids were co-\ncultured with NK cells as described above, they were \nthen resuspended into single cells using trypsin, pipet -\nted onto a droplet of Histogel (Epredia, MI, USA, #HG- \nID=\"EN6\">4000-012), then smeared onto a microscope \nslide. After the gel was air dried, cells were fixed with 4% \nparaformaldehyde for 30 min, permeabilised with 0.1% \nTriton X-100 for 10 min, then blocked with 1% BSA for \n2 h. Cells were then incubated first with anti-Ki-67 rab -\nbit antibodies (Abcam, Cambridge, UK, #ab16667; 1:250 \ndilution in 1% BSA) or rabbit IgG (Dako, #X0936; diluted \nto 4 ug/ml in 1% BSA) at 4 °C overnight, then with rabbit-\nanti mouse Alexa Fluor 568 antibodies (Thermo Fisher \nScientific, #a10042; 1:200 dilution in 1% BSA) for 2 h. \nNuclei was counterstained for 10 min with DAPI (Sigma \nAldrich, #D9542; diluted in PBS to 0.5 µg/ml). A drop of \nfluorescent mounting agent (Dako, #S3023) was added \nto the slide, a coverslip was mounted, and cells were \nanalysed using a BX60 fluorescent microscope (Olympus, \nJapan). The percentage of Ki67 positive cells over total \nnumber of live cells were calculated.\nStatistical analyses\nGraphPad Prism version 10 (San Diego, CA) was used \nfor statistical analysis. Paired t-test was applied wherever \nappropriate, and data were expressed as mean ± standard \ndeviation (SD); P ≤ 0.05 was considered significant.\nResults\nPODXL-KO Kuramochi spheroids are more vulnerable to NK \ncell infiltration than controls\nWe first employed spheroids formed with the Kuramo -\nchi line as a model of HGSC cells [ 24]. To study how \nPODXL levels may influence spheroid susceptibility to \nNK cells, we compared spheroids formed with control \nand PODXL-KO Kuramochi cells that we have previ -\nously engineered [ 24]. These spheroids were co-cultured \nwith human NK cells, which were isolated from PBMCs \nof healthy female donors ( n = 4) and labelled with CFSE \nfluorescent dye. The co-culture was examined after 24, 48 \nand 72 h. Representative brightfield and confocal images \nof spheroids immediately after the co-culture are shown \nin Fig. 1A. For both types of spheroids, a “ring” of NK \ncells was present around each spheroid, which was more \nobvious on the confocal image (Fig. 1A-a and b, both \npanels). The “thickness” of these “NK cell rings” around \ndid not change significantly over time in control spher -\noids (Fig. 1A-b, top panel), but they became increasingly \nthick and more dispersed towards the spheroid centre \nwith increasing time in co-culture in the PODXL-KO \nspheroids (Fig. 1A-b, bottom panel). When these co-\ncultured spheroids were washed with PBS to remove un-\ninfiltrated NK cells that surrounded the spheroids, the \nsize of control and PODXL-KO spheroids also appeared \nto differ (Fig. 1A-c, both panels). We thus further exam -\nined NK cell infiltration into spheroids and the impact \nof the co-culture on spheroid size and cell number. To \nanalyse NK cell infiltration, we determined the total fluo -\nrescence readings of NK cells that were contained inside \nthe spheroid after the co-culture but before PBS wash, \nreasoning that the wash might cause some NK cells to \nleak out of the spheroids. To achieve this, the outline of \neach spheroid was determined on the washed spheroid \nthen overlayed onto the confocal image of the un-washed \nspheroid (Fig. 1B, top panel), and the total NK cell fluo -\nrescence within the spheroid outlines were quantified \n(Fig. 1B, bar graph). While no difference was apparent \nat 24 h, significantly more NK cells were present inside \nthe PODXL-KO spheroids as compared to the control \nspheroids at both 48 h and 72 h of co-culture, indicating \nPODXL-KO spheroids sustained more NK cell infiltra -\ntion (Fig. 1B). \n\nPage 5 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nTo examine the impact of NK cell infiltration on spher-\noid size, we used the images of the washed spheroids (Fig. \n1C, images also show spheroid outlines used in Fig. 1B) \nand measured the diameters of these outlines and calcu -\nlated the spheroid volume (Fig. 1C, bar graph). No differ-\nence was obvious at 24 h, but at later timepoints control \nspheroids appeared to increase in size while PODXL-KO \nspheroid did not (Fig. 1C, bar graph). Consequently, the \nPODXL-KO spheroids were significantly smaller than \nthat of control spheroids at 72 h (Fig. 1C, bar graph). In \nthe absence of NK cells, at each time point, spheroids of \nboth the control and PODXL-KO did not differ much in \nsize (Supplementary Fig. 2 A).\nWe further analysed the total live cell numbers remain-\ning inside the spheroids (Fig. 1D). Within the first 24 h \nof NK cell co-culture, both spheroids showed a steep \ndecline in cell counts. However thereafter, cell numbers \ngradually bounced back in control spheroids but declined \nin PODXL-KO spheroids, resulting in significantly more \ncells in control as compared to PODXL-KO spheroids at \nboth 48 and 72 h (Fig. 1D). These data are consistent with \nthe spheroid size differences shown in Fig. 1C.\nPODXL-KO spheroids show higher NK cell-induced \ncytotoxicity than controls\nNext, we assessed how NK cell-induced cytotoxicity \ndiffered between PODXL-KO spheroids and controls. \nSpheroids were co-cultured with CFSE-labelled NK \ncells for 24, 48 and 72 h respectively as above, then cas -\npase-3/7 activity within spheroids was measured (Fig. \n2). Caspase-3/7 activity was apparent in both types of \nspheroids, but the signal was stronger in PODXL-KO \nas compared to control spheroids (Fig. 2A-b and c, both \npanels). Furthermore, more overlap was seen between \ncaspase-3/7 activity (red) with NK cell location (green) \ninside the spheroid in PODXL-KO (Fig. 2A-b, bottom \npanel) as compared control spheroids (Fig. 2A-b, top \npanel).\nTo further analyse the caspase-3/7 activity inside \nthe spheroids, confocal images of caspase-3/7 activity \nshown in Fig. 2A was overlayed with spheroid outlines \nas done for Fig. 1C, which indicated that the red signals \nwere contained inside the spheroids but more intensely \nin PODXL-KO compared to control spheroids (Fig. 2B). \nWe then measured the total red fluorescence readings \nFig. 1 Co-culture of control and PODXL-KO Kuramochi spheroids with human NK cells. A Representative images of spheroids co-cultured with NK cells \nfor 24, 48 and 72 h respectively. Top panel: control spheroids. Bottom panel: PODXL-KO (KO) spheroids. For each panel: a and b, immediately after the co-\nculture; c, after PBS wash to remove NK cells still present outside the spheroids; a and c, brightfield; b, confocal images of NK cells (fluorescently stained in \ngreen). B Analysis of NK cell infiltration into spheroids. Images of b in A) (NK cells in green) overlaid with the outlines of spheroids as yellow circles derived \nfrom images of c in A). Bar graph, total NK cell fluorescence present inside the spheroids. C Analysis of spheroid size following PBS wash. Images of c in \nA) presented together with yellow circle outlines shown in B). Bar graph, spheroid volume. D Analysis of live cells present within the spheroid after the \nwash. Data presented as percentage of live cells of untreated counterpart spheroids at each time point. Scale bar: 50 µm. Data as mean ± SD, n=4. *P < \n0.05, ** P<0.01, ****P < 0.0001\n \n\nPage 6 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \ninside these spheroids using a plate reader (Fig. 2C). In \nthe absence of NK cells, caspase-3/7 was negligible in the \nspheroids of both groups (Supplementary Fig. 2B). When \nco-cultured with NK cells, no difference was seen at 24 \nh, however with increasing time, the caspase-3/7 activ -\nity remained relatively stable in PODXL-KO spheroids \nbut decreased in control spheroids. As a result, PODXL-\nKO spheroids displayed significantly higher caspase-3/7 \nactivity than control spheroids, especially at 72 h (Fig. \n2C). These results indicate that PODXL-KO spheroids \nendured higher rates of NK cell-induced cytotoxicity \novertime than control spheroids.\nWe next examined the proliferative capacity of the sur -\nviving cells within the spheroids by assessing prolifera -\ntion marker Ki67 at 48 h after co-culture with NK cells \n(Fig. 3). The control spheroids displayed more Ki67-pos -\nitive cells than PODXL-KO spheroids (Fig. 3A). Quanti-\nfication showed that 56% of the cells in the control but \nonly 32% of the PODXL-KO spheroids were Ki67 positive \n(Fig. 3B). In the absence of NK cells, spheroids of both \ngroups consistently showed over 70–90% Ki67 positiv -\nity (Supplementary Fig. 2 C). Thus, Ki67 positivity was \ninversely correlated to caspase-3/7 activities in these \nspheroids (Figs. 2 and 3).\nMore NK cells infiltrate into primary HGSC spheroids that \nexpress lower levels of PODXL\nWe next investigated spheroids of ascites-derived pri -\nmary cells obtained from HGSC patients. Due to the \ndifficulty of culturing primary cells, we focused on cells \nfrom two patients that we previously found to express \nthe highest and lowest levels of PODXL among a cohort \n[24], and named them as high-PODXL and low-PODXL \ncells [#1 and #6 respectively as shown in [ 24]. Spheroids \nformed with these primary cells were co-cultured with \nNK cells for 24, 48 and 72 h and analysed as for Kura -\nmochi spheroids. Representative brightfield and confocal \nimages of these spheroids immediately after the NK cell \nco-culture are shown in Fig. 4A. Here, grossly NK cells \npenetrated more into the centre of both high-PODXL \nand low-PODXL spheroids (Fig. 4A-a and b, both pan -\nels), rather than forming “rings” as seen in Kuramochi \nspheroids (Fig. 1A).\nFig. 2  Analysis of caspase-3/7 activity within control and PODXL-KO \nKuramochi spheroids following co-culture with human NK cells. A Rep -\nresentative images of spheroids co-cultured with NK cells for 24, 48 and \n72 h respectively then analysed for caspase-3/7 activity. Top panel: con -\ntrol spheroids. Bottom panel: PODXL-KO (KO) spheroids. For each panel: a, \nbrightfield images of spheroids co-cultured with NK cells; b and c, confo -\ncal imaging of caspase-3/7 activity (red) overlaid with (b) or without (c) \nNK cells (green). B and C Analysis of caspase-3/7 activity. B) Images of c \nin A) overlaid with spheroid outlines as yellow circles. C) Quantification of \ncaspase-3/7 activity. Data presented as total fluorescence reading. Mean \n± SD, n=4. ** P<0.01\n \n\nPage 7 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nWhen NK cell infiltration was examined more closely \nas done for Kuramochi spheroids (Fig. 4B), no differ -\nence was seen at 24 h; however, with increasing co-cul -\nture time, the total NK cell fluorescence intensity inside \nthe spheroids decreased in high-PODXL but not in \nlow-PODXL spheroids (Fig. 4B, bar graph), leading to \nsignificantly more NK cells present inside low-PODXL \nspheroids than high-PODXL spheroids especially at 72 h.\nIn the absence of NK cells, at each time point, spheroids \nof high-PODXL and low-PODXL primary cells did not \ndiffer much in size (Supplementary Fig. 3 A). In contrast, \nwhen co-cultured with NK cells, both groups showed \na trend of size reduction over time, but low-PODXL \nspheroids were significantly smaller than high-PODXL \nspheroids (Fig. 4C, bar graph). Counting live cells pres -\nent inside these spheroids revealed that for both high-\nPODXL and low-PODXL spheroids, the live cell numbers \nreduced sharply following the initial 24 h co-culture (Fig. \n4D). At later timepoints, cell numbers started to bounce \nback somewhat but the increase was greater in high-\nPODXL as compared to low-PODXL spheroids, resulting \nin more cells in high-PODXL than low-PODXL spher -\noids overall (Fig. 4D). Collectively, these data of primary \nHGSC spheroids are consistent with the Kuramochi \nspheroids, showing that lower PODXL expression cor -\nrelated with more NK cell infiltration and greater reduc -\ntions in spheroid size and live cell numbers.\nPrimary HGSC spheroids expressing lower PODXL display \nhigher NK cell-induced cytotoxicity\nWe also examined NK cell-induced cytotoxicity in pri -\nmary HGSC spheroids (Fig. 5). Primary spheroids were \nco-cultured with NK cells as above, then assessed for cas-\npase-3/7 activity. Due to limited availability of primary \ncells, and because studies of caspase-3/7 activity with the \nKuramochi line showed little difference at 24 h (Fig. 2B \nand C), here we only examined the caspase-3/7 activity \nafter 48 and 72 h co-culture of primary spheroids with \nNK cells (Fig. 5).\nCaspase-3/7 activities were present in both groups \nof spheroids, however the overlap between NK cells \nlocation and caspase-3/7 activity appeared to be more \ntowards the centre in low-PODXL than high-PODXL \nspheroids (Fig. 5A). In the absence of NK cells, cas -\npase-3/7 was negligible in the spheroids of both groups \n(Supplementary Fig. 3B). The Closer examination of cas -\npase-3/7 fluorescence presentation showed, visually (Fig. \n5B) and quantitatively (Fig. 5C), that caspase-3/7 activity \nwas higher in low-PODXL as compared to high-PODXL \nspheroids at both 48 h and 72 h.\nWe also assessed the proliferative capacity of the sur -\nviving cells within these primary spheroids by staining \nfor Ki67 at 48 h (Fig. 6). Visually (Fig. 6A) and quanti -\ntively (Fig. 6B), fewer Ki67-positive cells were present \nin low-PODXL as compared to high-PODXL spheroids. \nTogether, these results suggest an inverse correlation \nbetween PODXL levels and NK cell-induced cytotoxic -\nity, consistent with the data observed with Kuramochi \nspheroids.\nDiscussion\nThis study investigated whether PODXL levels in HGSC \nspheroids may influence NK cell infiltration and cytotox -\nicity. We first used a cell line model, employing spheroids \nformed with control and PODXL-KO Kuramochi cells. \nWhen these spheroids were co-cultured with human \nNK cells, PODXL-KO spheroids showed higher NK cell \ninfiltration and greater NK cell-induced cytotoxicity \nthan controls spheroids, resulting in smaller spheroids \nwith fewer live and proliferative cells remaining in the \nformer compared to the latter. We then validated the \ndata in primary spheroids derived from ascites of HGSC \npatients expressing different levels of PODXL. Again, NK \ncell infiltration was greater and more severe cytotoxic -\nity in primary HGSC spheroids that expressed lower \nFig. 3 Analysis of cell proliferation marker Ki67 in Kuramochi spheroids \nfollowing co-culture with NK cells. Data of control and PODXL-KO (KO) \nspheroids co-cultured with NK cells for 48 h are presented. A Representa-\ntive images of Ki67 immunostaining (red); blue, DAPI. Scale bar: 50 µm. \nB Quantification of Ki67 staining. Data presented as percentage of Ki67-\npositive cells over all live cells. Mean ± SD, n=4. ** P<0.01\n \n\nPage 8 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nthan higher levels of PODXL, leading to worse spheroid \ndestruction and less proliferative capacity in the former. \nCollectively, these data suggest that PODXL may play an \nimportant protective role in HGSC spheroids from NK \ncell infiltration and spheroid destruction.\nSeveral prior studies have demonstrated that NK cells \ncan destroy cancer cells when added to cell monolayers \n[11, 12, 30]. However, only a few studies have investigated \nthe effects of NK cells using spheroid models which can \nmimic the in vivo cell architecture, morphology and cell-\ncell interactions of metastatic spheroids [ 31– 36]. In par -\nticular, NK cells derived from human hematopoietic stem \nand progenitor cells were reported to actively infiltrate \nand kill OC spheroids formed with cell lines SKOV3, \nIGROV1, and OVCAR3 in a dose dependent manner \n[36]. They are consistent with our findings that NK cells \ncan infiltrate and destroy HGSC spheroids. However, \nour studies considerably extended this line of research \nand investigated the potential role of a specific molecule, \nPODXL, whose expression has been positively associated \nwith poor prognosis of HGSC patients.\nIn Kuramochi spheroids, the levels of PODXL made a \nsignificant difference in spheroid susceptibility to NK cell \ninfiltration and spheroid destruction. We saw more NK \ncell penetration, bigger drops in spheroid volume, and \na larger reduction in live cell numbers in PODXL-KO \nspheroids than the control over times during co-culture \nwith NK cells. As NK cell infiltration has been reported \nto be associated with cancer cell apoptosis inside the \nspheroid [31, 32, 36], we also examined apoptosis using \ncaspase-3/7 activity as a marker. Indeed, we observed \nhigher caspase-3/7 activities in PODXL-KO spheroids \nthan controls, which positively correlated with their dif -\nferences in NK cell penetration. Further analysis of prolif-\nerative capacity of cells remaining in the spheroid showed \nthat more Ki67-positive cells were present in the control \nthan PODXL-KO spheroids, indicating that the control \nspheroids are more actively proliferating and recover -\ning following NK cell attack than PODXL-KO spheroids. \nSimilar observations were also made with ascites-derived \nprimary HGSC spheroids following co-culture with NK \ncells; spheroids expressing lower PODXL exhibited more \nNK cell infiltration, higher apoptosis and less prolifera -\ntion than spheroids with higher PODXL. To our knowl -\nedge, this is the first study to utilise ascites-derived \npatient cancer spheroids to examine NK cell infiltration \nas well as the importance of PODXL. A recent study has \nreported that a glycopeptide epitope on the extracellular \ndomain of PODXL, which is expressed only on cancer \ncells, is correlated with poor immune cell infiltration in \nFig. 4 Co-culture of ascites-derived primary HGSC spheroids with human NK cells. A Representative images of spheroids of HGSC cells expressing high \n(High-PODXL) or low (Low-PODXL) levels of PODXL that were co-cultured with NK cells for 24, 48 and 72 h respectively. Top panel: High-PODXL spheroids. \nBottom panel: Low-PODXL spheroids. For each panel: a and b, immediately after the co-culture; c, after PBS wash to remove NK cells still present outside \nthe spheroids; a and c, brightfield; b, confocal images of NK cells (fluorescently stained in green). B Analysis of NK cell infiltration into spheroids. Images \nof b in A) (NK cells in green) overlaid with spheroid outlines as yellow circles derived from images of c in A). Bar graph, total NK cell fluorescence present \ninside the spheroids. C Analysis of spheroid size following the PBS wash. Images of c in A) are presented together with yellow circle outlines shown in B). \nBar graph, spheroid volume. D Analysis of live cells present within the spheroid after the wash. Data presented as percentage of live cells of the untreated \ncounterpart spheroid at each time point. Scale bar: 50 µm. Data as Mean ± SD, n=4. *P < 0.05, ** P<0.01\n \n\nPage 9 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nHGSC tumors [ 37]. Although it is unclear how this gly -\ncopeptide epitope dictates immune infiltration, this study \nlargely supports our investigation that PODXL may play \na role in impeding immune cell penetration and thus, \nremoval of PODXL may present a treatment opportunity \nto increase anti-cancer immune responses in HGSC [ 38, \n39].\nNK cell receptors recognise and destroy malignant cells \nthrough activating or inhibitory signals to induce cyto -\ntoxicity [40]. As an immune evasion strategy, cancer cells \ncan modulate the expression of corresponding activating \nor inhibitory ligands to escape NK cell attack [ 41]. Stud-\nies in MCF7 breast cancer cells have demonstrated that \nPODXL expression on cancer cells can downregulate the \nNK cell activating receptors such as NKG2D, NKp30, and \nNKp44 to influence NK cell activity [ 42]. Many cancers \nare also known to shed NKG2D ligands as part of the \ntumor immune evasion strategy [ 43]. In a study using \nCaSki and SiHa cervical cancer cell line spheroids, co-\nculture with NK cells showed an accumulation of sol -\nuble NKG2D ligands (MICA, MICB and ULBP2) in the \nsupernatant of co-cultures, which paralleled the loss of \nligands from the cell surface [ 31]. This suggests that can-\ncer spheroids can continuously shed cellular ligands of \nNKG2D [31]. We therefore speculate that PODXL could \nplay a role in evasion of NK cell killing through facilitat -\ning the shedding of NK cell activating ligands on HGSC \ncells, although this needs further investigation.\nIt is well known that age is a strong risk factor of OC \nespecially in those over the age of 65 [44], however, mech-\nanisms remain unclear. Since physiological aging of NK \ncells are known to be associated with NK cell immunose -\nnescence, a phenomenon that is linked to decreased NK \ncell activity and increased incidences of infections [45], in \nthis study we used NK cells of young (aged 21–28) and \nhealthy females to ensure their activity was not compro -\nmised by age. Future studies are warranted to investigate \nwhether the age of NK cells also matters, and whether \nolder women have physiologically older NK cells that \nare less potent for control of cancer cells. If this is true, it \nmay provide novel insights into the understanding of why \nage increases the risk of OC. It would also rationalise why \nFig. 5  Analysis of caspase-3/7 activity following co-culture of ascites-\nderived primary HGSC spheroids with NK cells. A Representative images \nof spheroids expressing high (High-PODXL) or low (Low-PODXL) levels \nof PODXL co-cultured with NK cells for 24, 48 and 72 h respectively then \nanalysed for caspase-3/7 activity. Top panel: High-PODXL spheroids. Bot -\ntom panel: Low-PODXL spheroids. For each panel: a, brightfield images \nof spheroids co-cultured with NK cells; b and c, confocal imaging of cas -\npase-3/7 activity (red) overlaid with (b) or without (c) NK cells (green). B \nand C Analysis of caspase-3/7 activity. B) Images of c in A) overlaid with \noutlines of spheroids as yellow circles. C) Quantification of caspase-3/7 \nactivity. Data presented as total fluorescence reading. Mean ± SD, n=4. \n*P < 0.05\n \n\nPage 10 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nFig. 6 Analysis of cell proliferation marker Ki67 in primary cancer spheroids following co-culture with NK cells. Data of spheroids of expressing high \n(High-PODXL) or low (Low-PODXL) levels of PODXL co-cultured with NK cells for 48 h are presented. A Representative images of Ki67 immunostaining \n(red); blue, DAPI. Scale bar: 50 µm. B Quantification of Ki67 staining. Data presented as percentage of Ki67-positive cells over all live cells. Mean ± SD, n=4. \n**** P<0.0001\n \n\nPage 11 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \nautologous NK cell therapy can be less effective [ 46, 47], \nas allogeneic NK cells from younger donors may lead to \nmore favourable outcomes in older patients.\nA limitation of this study was the limited amount of \nascites-derived primary cells that we were able to acquire \nand maintain in culture. Furthermore, as HGSC is a het -\nerogenous disease, the physiological differences observed \nin the primary cells may be influenced by other factors \ndue to patient variation. Therefore, further studies with \na larger cohort of patients would help further establish \nthe correlations between PODXL expression and patient \nresponse to NK cell infiltration and cytotoxicity.\nIn summary, our data suggests that PODXL plays an \nimportant, protective role in hindering NK cell infiltra -\ntion and NK cell-mediated destruction of HGSC cancer \nspheroids, and that lowering the levels of PODXL may \nsensitise HGSC to NK cells. Consequently, strategies \nto downregulate PODXL in HGSC patients with high \nPODXL expression may assist them to fight the cancer \nthrough their own NK cells and/or through adoptive \nimmunotherapy using donor NK cells.\nAbbreviations\nOC  Ovarian cancer\nHGSC  High grade serous carcinoma\nNK  Natural killer\nPODXL  Podocalyxin\nPBMCs  Peripheral blood mononuclear cells\nKO  Knockout\nCFSE  Carboxyfluoroscein succinimidyl ester\nSupplementary Information\nThe online version contains supplementary material available at  h t t p  s : /  / d o i  . o  r \ng /  1 0 .  1 1 8 6  / s  1 2 8 8 5 - 0 2 5 - 1 5 1 0 8 - 6.\nSupplementary Material 1.\nSupplementary Material 2.\nSupplementary Material 3.\nSupplementary Material 4.\nAcknowledgements\nNot applicable.\nAuthors’ contributions\nG.N conceived and oversaw the project, G.N and Y.W and N.L.T designed \nthe study. N.L.T conducted the experiments, analysed the data, and wrote \nthe manuscript under the guidance of Y.W and G.N. K.Q. provided the blood \nsamples and guidance in PBMC isolation. M.B. and A.S. provided ascites-\nderived primary cells. All authors reviewed the manuscript.\nFunding\nThis study was supported by the National Health and Medical Research \nCouncil (NHMRC) of Australia (#2012523 to G.N) and Contributing to \nAustralian Scholarship and Science (CASS) foundation (#10453 to Y.W).\nData availability\nThe datasets used and/or analysed during the current study are available from \nthe corresponding author on reasonable request.\nDeclarations\nEthics approval and consent to participate\nStudy was provided by the Australian Red Cross with ethics approval by RMIT \nCollege of Human Ethics Advisory Network (#28056). All work was conducted \naccording to the Declaration of Helsinki Principles and the Australian National \nHealth and Medical Research Council (NHMRC) Code of Practice. Signed \ninformed consent was obtained from all donors before the study.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare no competing interests.\nReceived: 30 June 2025 / Accepted: 26 September 2025\nReferences\n1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer \nstatistics 2018: GLOBOCAN estimates of incidence and mortality worldwide \nfor 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424.\n2. Franier B, Thompson M. Early stage detection and screening of ovarian \ncancer: A research opportunity and significant challenge for biosensor tech-\nnology. Biosens Bioelectron. 2019;135:71–81.\n3. Colombo N, Sessa C, Bois AD, Ledermann J, McCluggage WG, McNeish I, et \nal. ESMO-ESGO consensus conference recommendations on ovarian cancer: \npathology and molecular biology, early and advanced stages, borderline \ntumours and recurrent disease. Int J Gynecol Cancer. 2019.  h t t p s :   /  / d o  i .  o r  g  /  1 0  . \n1 1   3 6  / i  j g c - 2  0 1 9 - 0 0 0 3 0 8.\n4. Markman M, Markman J, Webster K, Zanotti K, Kulp B, Peterson G, et al. Dura-\ntion of response to second-line, platinum-based chemotherapy for ovarian \ncancer: implications for patient management and clinical trial design. J Clin \nOncol. 2004;22(15):3120–5.\n5. Ledermann JA, Raja FA, Fotopoulou C, Gonzalez-Martin A, Colombo N, Sessa \nC, et al. Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO \nclinical practice guidelines for diagnosis, treatment and follow-up. Ann \nOncol. 2013;24(Suppl 6):vi24–32.\n6. Naora H, Montell DJ. Ovarian cancer metastasis: integrating insights from \ndisparate model organisms. Nat Rev Cancer. 2005;5(5):355–66.\n7. Ahmed N, Stenvers KL. Getting to know ovarian cancer ascites: opportunities \nfor targeted therapy-based translational research. Front Oncol. 2013;3:256.\n8. Galon J, Pages F, Marincola FM, Angell HK, Thurin M, Lugli A, et al. Cancer \nclassification using the immunoscore: a worldwide task force. J Transl Med. \n2012;10:205.\n9. Hao J, Li M, Zhang T, Yu H, Liu Y, Xue Y, et al. Prognostic value of Tumor-\nInfiltrating lymphocytes differs depending on lymphocyte subsets in \nesophageal squamous cell carcinoma: an updated Meta-Analysis. Front \nOncol. 2020;10:614.\n10. Webb JR, Milne K, Watson P , Deleeuw RJ, Nelson BH. Tumor-infiltrating \nlymphocytes expressing the tissue resident memory marker CD103 are \nassociated with increased survival in high-grade serous ovarian cancer. Clin \nCancer Res. 2014;20(2):434–44.\n11. Geller MA, Knorr DA, Hermanson DA, Pribyl L, Bendzick L, McCullar V, et al. \nIntraperitoneal delivery of human natural killer cells for treatment of ovarian \ncancer in a mouse xenograft model. Cytotherapy. 2013;15(10):1297–306.\n12. Hermanson DL, Bendzick L, Pribyl L, McCullar V, Vogel RI, Miller JS, et al. \nInduced pluripotent stem Cell-Derived natural killer cells for treatment of \novarian cancer. Stem Cells. 2016;34(1):93–101.\n13. Uppendahl LD, Dahl CM, Miller JS, Felices M, Geller MA. Natural killer Cell-\nBased immunotherapy in gynecologic malignancy: A review. Front Immunol. \n2017;8:1825.\n14. Chiossone L, Dumas PY, Vienne M, Vivier E. Natural killer cells and other innate \nlymphoid cells in cancer. Nat Rev Immunol. 2018;18(11):671–88.\n15. Hoogstad-van Evert JS, Maas RJ, van der Meer J, Cany J, van der Steen S, Jan-\nsen JH, et al. Peritoneal NK cells are responsive to IL-15 and percentages are \ncorrelated with outcome in advanced ovarian cancer patients. Oncotarget. \n2018;9(78):34810–20.\n\nPage 12 of 12\nTran et al. BMC Cancer         (2025) 25:1674 \n16. Sconocchia G, Eppenberger S, Spagnoli GC, Tornillo L, Droeser R, Caratelli S, \net al. NK cells and T cells cooperate during the clinical course of colorectal \ncancer. Oncoimmunology. 2014;3(8):e952197.\n17. Marin D, Li Y, Basar R, Rafei H, Daher M, Dou J, et al. Safety, efficacy and deter-\nminants of response of allogeneic CD19-specific CAR-NK cells in CD19(+) B \ncell tumors: a phase 1/2 trial. Nat Med. 2024;30(3):772–84.\n18. Lanier LL. NK cell recognition. Annu Rev Immunol. 2005;23:225–74.\n19. Cheng M, Chen Y, Xiao W, Sun R, Tian Z. NK cell-based immunotherapy for \nmalignant diseases. Cell Mol Immunol. 2013;10(3):230–52.\n20. Caligiuri MA. Human natural killer cells. Blood. 2008;112(3):461–9.\n21. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. \nInnate or adaptive immunity? The example of natural killer cells. Science. \n2011;331(6013):44–9.\n22. Motohara T, Masuda K, Morotti M, Zheng Y, El-Sahhar S, Chong KY, et al. An \nevolving story of the metastatic voyage of ovarian cancer cells: cellular and \nmolecular orchestration of the adipose-rich metastatic microenvironment. \nOncogene. 2019;38(16):2885–98.\n23. Le Tran N, Wang Y, Nie G. Podocalyxin in normal tissue and epithelial cancer. \nCancers (Basel). 2021;13(12).\n24. Le Tran N, Wang Y, Bilandzic M, Stephens A, Nie GY. Podocalyxin promotes the \nformation of compact and chemoresistant cancer spheroids in high grade \nserous carcinoma. Sci Rep-Uk. 2024;14(1).\n25. Cipollone JA, Graves ML, Kobel M, Kalloger SE, Poon T, Gilks CB, et al. The \nanti-adhesive mucin podocalyxin May help initiate the transperitoneal \nmetastasis of high grade serous ovarian carcinoma. Clin Exp Metastasis. \n2012;29(3):239–52.\n26. Ney JT, Zhou H, Sipos B, Buttner R, Chen X, Kloppel G, et al. Podocalyxin-like \nprotein 1 expression is useful to differentiate pancreatic ductal adenocarci-\nnomas from adenocarcinomas of the biliary and Gastrointestinal tracts. Hum \nPathol. 2007;38(2):359–64.\n27. Somasiri A, Nielsen JS, Makretsov N, McCoy ML, Prentice L, Gilks CB, et al. \nOverexpression of the anti-adhesin podocalyxin is an independent predictor \nof breast cancer progression. Cancer Res. 2004;64(15):5068–73.\n28. Le Tran N, Wang Y, Bilandzic M, Stephens A, Nie G. Podocalyxin promotes the \nformation of compact and chemoresistant cancer spheroids in high grade \nserous carcinoma. Sci Rep. 2024;14(1):7539.\n29. Heng S, Samarajeewa N, Wang Y, Paule SG, Breen J, Nie G. Podocalyxin \npromotes an impermeable epithelium and inhibits pro-implantation factors \nto negatively regulate endometrial receptivity. Sci Rep. 2021;11(1):24016.\n30. Bhat R, Watzl C. Serial killing of tumor cells by human natural killer cells–\nenhancement by therapeutic antibodies. PLoS ONE. 2007;2(3):e326.\n31. Giannattasio A, Weil S, Kloess S, Ansari N, Stelzer EH, Cerwenka A, et al. \nCytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids. \nBMC Cancer. 2015;15:351.\n32. Courau T, Bonnereau J, Chicoteau J, Bottois H, Remark R, Assante Miranda L, \net al. Cocultures of human colorectal tumor spheroids with immune cells \nreveal the therapeutic potential of MICA/B and NKG2A targeting for cancer \ntreatment. J Immunother Cancer. 2019;7(1):74.\n33. Lanuza PM, Vigueras A, Olivan S, Prats AC, Costas S, Llamazares G, et al. \nActivated human primary NK cells efficiently kill colorectal cancer cells in 3D \nspheroid cultures irrespectively of the level of PD-L1 expression. Oncoimmu-\nnology. 2018;7(4):e1395123.\n34. Morimoto T, Nakazawa T, Matsuda R, Nishimura F, Nakamura M, Yamada S, et \nal. Evaluation of comprehensive gene expression and NK cell-Mediated killing \nin glioblastoma cell Line-Derived spheroids. Cancers (Basel). 2021;13:19.\n35. Varudkar N, Oyer JL, Copik A, Parks GD. Oncolytic parainfluenza virus \ncombines with NK cells to mediate killing of infected and non-infected lung \ncancer cells within 3D spheroids: role of type I and type III interferon signal-\ning. J Immunother Cancer. 2021;9(6).\n36. Hoogstad-van Evert JS, Cany J, van den Brand D, Oudenampsen M, Brock R, \nTorensma R et al. Umbilical cord blood CD34 progenitor-derived NK cells \nefficiently kill ovarian cancer spheroids and intraperitoneal tumors in NOD/\nSCID/IL2Rg mice. Oncoimmunology. 2017;6(8).\n37. Brassard J, Hughes MR, Dean P , Hernaez DC, Thornton S, Banville AC, et al. \nA tumor-restricted glycoform of podocalyxin is a highly selective marker of \nimmunologically cold high-grade serous ovarian carcinoma. Front Oncol. \n2023;13:1286754.\n38. Milne K, Kobel M, Kalloger SE, Barnes RO, Gao D, Gilks CB, et al. System-\natic analysis of immune infiltrates in high-grade serous ovarian cancer \nreveals CD20, FoxP3 and TIA-1 as positive prognostic factors. PLoS ONE. \n2009;4(7):e6412.\n39. Nielsen JS, Sahota RA, Milne K, Kost SE, Nesslinger NJ, Watson PH, et al. \nCD20 + tumor-infiltrating lymphocytes have an atypical CD27- memory \nphenotype and together with CD8 + T cells promote favorable prognosis in \novarian cancer. Clin Cancer Res. 2012;18(12):3281–92.\n40. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer \ncells. Nat Immunol. 2008;9(5):503–10.\n41. Raulet DH, Gasser S, Gowen BG, Deng W, Jung H. Regulation of ligands for \nthe NKG2D activating receptor. Annu Rev Immunol. 2013;31:413–41.\n42. Amo L, Tamayo-Orbegozo E, Maruri N, Buque A, Solaun M, Rinon M, et al. \nPodocalyxin-like protein 1 functions as an Immunomodulatory molecule in \nbreast cancer cells. Cancer Lett. 2015;368(1):26–35.\n43. Salih HR, Holdenrieder S, Steinle A. Soluble NKG2D ligands: prevalence, \nrelease, and functional impact. Front Biosci. 2008;13:3448–56.\n44. Australian Institute of H, Welfare. Cancer in Australia: Actual incidence data \nfrom 1982 to 2013 and mortality data from 1982 to 2014 with projections to \n2017. Asia Pac J Clin Oncol. 2018;14(1):5–15. \n45. Solana R, Alonso MC, Peña J. Natural killer cells in healthy aging. Exp Gerontol. \n1999;34(3):435–43.\n46. Parkhurst MR, Riley JP , Dudley ME, Rosenberg SA. Adoptive transfer of \nautologous natural killer cells leads to high levels of Circulating natu-\nral killer cells but does not mediate tumor regression. Clin Cancer Res. \n2011;17(19):6287–97.\n47. Nersesian S, Glazebrook H, Toulany J, Grantham SR, Boudreau JE. Naturally \nkilling the silent killer: NK Cell-Based immunotherapy for ovarian cancer. Front \nImmunol. 2019;10:1782.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in \npublished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}