Vimentin in the tumor microenvironment: orchestrating invasion, immunity, and metabolism.

Transcriptional control of epithelial to mesenchymal transition by regulatory factors and epigenetic mechanisms

Editor's evaluation: Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Decision letter: Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on chimeric antigen receptor modified T cell (CAR-T) immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase and programmed cell death-ligand 1 which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial reactive oxygen species release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the stanniocalcin-1 (STC1) gene, which encodes the glycoprotein hormone STC-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo, and STC1 played a critical role. These data revealed a novel function of MSC and STC-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response. Editor's evaluation This study uncovers the contributions of MSC on modulating CAR T-cell behaviour. Based on the importance in basic biology and its immediate impact on translational potential, all reviewers are satisfied on the advances in this study. https://doi.org/10.7554/eLife.82934.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Immunotherapy is a type of cancer treatment that helps the immune system fight cancer. For example, chimeric antigen receptor T cell (CAR-T) therapy is used to target several types of blood cancer. It works by reprogramming patients’ immune cells to target specific tumor cells. In blood cancers, CAR-T therapy works very well, but it can cause extreme responses from the patient’s immune system, which can be life threatening. In solid tumors, CAR-T therapy is much less successful because the tumors secrete molecules into the space surrounding them, which weaken the immune processes that attack cancerous cells. Stem cells are the master cells of the body. Originating in the bone marrow, they can repair and regenerate the body’s cells. Cancer stem cells play a role in resistance to CAR-T therapy, due – in part – to their ability to renew themselves, but the role of another type of stem cell, called mesenchymal stem cells, was less clear. Mesenchymal stem cells develop into tissues that line organs and blood vessels. Although it is known that mesenchymal stem cells are present in most cancers and play a role in shaping and influencing the space around tumors, their impact on CAR-T therapy has not been studied in depth. To find out more, Zhang et al. looked at the influence of a protein, called staniocalcin-1 (STC1), on CAR-T therapy, by studying cells grown in the laboratory and human tumor cells that had been implanted in mice. Zhang et al. found that mesenchymal stem cells reduce the ability of CAR-T therapy to destroy cancer cells and that they needed STC1 to do this successfully. They also increased the expression of molecules that dampen the immune system, and suppressed molecules called inflammasomes, which are an important part of the way the immune system detects disease. Moreover, reducing the amount of STC1 that mesenchymal stem cells expressed restored the effectivity of CAR-T therapy. This study increases our understanding of the way that mesenchymal stem cells affect CAR-T therapy. It has the potential to open up a new way of improving the efficiency of this treatment and of reducing the harmful side effects that it can cause. Introduction Advances in chimeric antigen receptor modified T cell therapy (CAR-T) in recent years have shown enormous promise in cancer immunotherapy, which has produced unprecedented clinical outcomes, most notably for patients with hematologic malignancies (Singh et al., 2016; Park et al., 2018). Despite the striking achievements, CAR-T therapy is also facing many challenges such as the treatment-related severe toxicity and side effects, including cytokine release syndrome (CRS) and neurotoxicity (Hong et al., 2020; Freyer and Porter, 2020). CRS is the most common acute toxicity associated with an excessive immune response that causes fever, hypotension, and respiratory insufficiency. The neurotoxicity induced by CAR-T therapy exhibits a diverse array of neurologic symptoms such as tremors, expressive aphasia, and impaired attention. The precise mechanism that causes these life-threatening side effects remains unclear (Freyer and Porter, 2020; Jiang et al., 2019). On the other hand, the success of CAR-T therapy in treating solid tumors is still very limited (Martinez and Moon, 2019). Identifying hurdles and potential mechanisms that impede the function of CAR-T cells is of vital importance to expanding its use. The immunosuppressive tumor microenvironment (TME) is one of the obstacles that diminishes the efficacy of CAR-T therapy, especially for solid tumors. Among the many factors that can modulate TME and immune response, the impact of mesenchymal stem cell (MSC) on CAR-T therapy has been little studied. MSC is a type of adult stem cell with high proliferative activity and multidirectional differentiation capacity. However, MSCs have additional paracrine effects that are believed to underlie their therapeutic functions (Jiang and Xu, 2020). By secreting a variety of cytokines into the tissue microenvironment, it has been known that MSCs can modulate extracellular matrix, promote angiogenesis, and suppress inflammation and apoptosis (Keating, 2012; Wang et al., 2014; Regmi et al., 2019). Some MSC-secreted cytokines, such as stromal cell-derived factor 1 and stem cell factor, play important roles in hematopoietic and immune regulation (Kawaguchi et al., 2019; Markov et al., 2007). In addition, studies suggest that MSCs can modulate the function of monocytic lineages cells, especially macrophages (Németh et al., 2009; YlÖstalo et al., 2012; Choi et al., 2011). Some reports also showed that MSCs could directly affect the functionality and cellular responses of T cells, Tregs, and memory T cells (Cen et al., 2019; Tumangelova-Yuzeir et al., 2019; Luque-Campos et al., 2019). It was reported that human mesenchymal stem cells (hMSCs) could be activated by lipopolysaccharide (LPS)-stimulated macrophages to increase the expression and secretion of stanniocalcin-1 (STC1) (Oh et al., 2014). STC1 was a mitochondria-related glycoprotein originally identified as a calcium/phosphate regulating hormone in bony fishes, and later on, it was found to be a pleiotropic factor involved in various degenerative diseases such as ocular and renal disease, as well as idiopathic pulmonary fibrosis (Yeung et al., 2012; Ohkouchi et al., 2015). STC1 could improve the cell survival and regeneration of MSCs in a paracrine fashion (Ono et al., 2015). There was also evidence suggesting that STC1 played an oncogenic role in various types of tumors (Du et al., 2011; Liu et al., 2010). Based on a retrospective study of ~1500 clinical samples, it was concluded that high STC1 expression is associated with the poor clinical outcome of breast cancer (Chang et al., 2015). It was proved that STC1 is involved in several oxidative and cancer-related signaling pathways, such as NF-κB, extracellular-signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) pathways (Nguyen et al., 2009; Chan et al., 2017). The expression and secretion of STC1 in cancer tissue can be stimulated by external stimuli, including external cytokines and oxidative stress (Nguyen et al., 2009). Under hypoxia conditions, STC1 could be modulated by Hypoxia-inducible factor-1 (HIF-1) to facilitate the reprogramming of tumor metabolism from oxidative to glycolytic metabolism (Yeung et al., 2005). STC1 was also reported to participate in the process of epithelial-to-mesenchymal transition, which is associated with tumor invasion and the reshape of the tumor microenvironment, as well as increasing therapy resistance (Pastushenko and Blanpain, 2019). Considering the pleiotropic role of STC1, especially its intercellular linkage between MSCs, cancer cells, and macrophage stimulation, it is interesting to know what role it plays in connection to the functions of MSC in TME. Therefore, we generated a stable STC1 knockdown MSC cell line. With a cell co-culture model containing CAR-T cells, hMSCs, macrophages, and Pfeiffer lymphoma cells to partially mimic the tumor microenvironment together with a xenograft mice model, here we studied the impacts of MSC on CAR-T efficacy and the potential immune response change in the presence and absence of STC1. Results Stable knockdown of STC1 in hMSC-inhibited cell migration, slightly suppressed cell proliferation, but no increase in apoptosis To study the function of STC1, we first generated a stable knockdown cell line by lentivirus-based shRNA for the STC1 gene, and the expression of STC1 protein was evaluated by Western blot (Figure 1A). STC1 stable knockdown in hMSCs exhibited a minor effect in cell survival (Figure 1B) and slightly reduced proliferation rate based on the small increase in the proportion of cells in G0/G1 phases versus that in the S phase (Figure 1C) as determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and Fluorescence-activated Cell Sorting (FACS) analysis. To investigate whether knockdown of STC1 affects cell migration, wound healing and transwell chamber assays were performed. After creating a ‘scratch’ in a monolayer of hMSCs, the closure of the gap was determined after 24 hr. As shown in Figure 1D, compared to control hMSCs, the gap was less filled in hMSCshSTC1. The inhibitory effect on cell migration was further confirmed by a transwell assay. As shown in Figure 1E, there were significant migration and invasion observed in hMSCsshCtrl, whereas there was a >30% reduction in migration across the transwell chamber membrane in hMSCsshSTC1. To further determine whether knockdown of STC1 may have any lethal effect, apoptosis was determined by two different assays. To measure the early apoptosis, cells were stained with the Alexa Fluor 488 annexin V and the propidium iodide (PI) followed by flow cytometry to detect apoptosis-associated phosphatidylserine (PS) expression and membrane permeability (Figure 1F). Parallelly, no DNA fragmentation was detected as determined with the TUNEL assay (Figure 1G, the green dots were from the background due to overexposure). Both studies showed that knockdown of STC1 did not cause apoptosis of hMSCs. Figure 1 Download asset Open asset The impact of stanniocalcin-1 (STC1) knockdown on cell proliferation, migration, and apoptosis of hMSCs. (A) Western blot analysis of STC1 protein expression in hMSCs. (B) Cell viability determined by MTT, measurements are shown as the mean ± SD from three independent experiments. (C) FACS analysis of cell cycle progression on hMSCs w/o STC1 knockdown. (D, E) Knockdown of STC1 suppressed cell migration as determined by wound healing and transwell chamber assays. (F) Apoptosis determination by the Alexa Fluor 488 annexin V and PI detection. (G) DNA fragmentation determination by transferase-mediated dUTP nick-end labeling (TUNEL) assay. Figure 1—source data 1 Labeled original blots of Figure 1A. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data1-v3.zip Download elife-82934-fig1-data1-v3.zip Figure 1—source data 2 Unlabeled original blots of Figure 1A. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data2-v3.zip Download elife-82934-fig1-data2-v3.zip Figure 1—source data 3 Figure 1B in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data3-v3.xlsx Download elife-82934-fig1-data3-v3.xlsx The presence of hMSCs inhibited CAR-T cell killing activity, but knockdown of STC1 completely abrogated this inhibition To investigate the impact of hMSCs on CAR-T treatment, we used an in vitro cell co-culture model modified according to previous studies to mimic a simplified situation of tumor environment (Singh et al., 2017; Liu et al., 2021). The co-culture contained CD19 CAR-T cells, Pfeiffer cells that were from human diffuse large cell lymphoma, and M2 macrophages (derived from THP-1 cells by phorbol-12-myristate-13-acetate [PMA] polarization for 24 hr) at a cell number ratio of 1:3:1. The cell-killing activity of CAR-T cells toward Pfeiffer cells was determined by lactate dehydrogenase (LDH) cytotoxicity assay on total cell co-culture. As shown in Figure 2A, 67% of Pfeiffer cells were killed after being exposed to CAR-T cells for 24 hr, and 93% were killed at 48 hr as compared to mock-treated control. After adding hMSCs into the co-culture, the cell-killing activity of CAR-T was significantly inhibited (Figure 2A). The number of hMSC added was the same as the CAR-T cell. Interestingly, the inhibitory effect of hMSCs on CAR-T cytotoxicity could be completely abrogated if knockdown STC1 gene in hMSCs. These results for the first time revealed that CAR-T efficacy could be affected by the presence of MSCs, and the gene STC1 played a critical role. Figure 2 Download asset Open asset Analysis of cytotoxicity, T cell composition, and immune-suppressive markers. The cell co-culture contained chimeric antigen receptor modified T cell (CAR-T) cells, Pfeiffer cells, M2 macrophages, and control or stanniocalcin-1 (STC1) knockdown hMSCs in a ratio of 1:3:1:1. After 24 hr (or 48 hr for cytotoxicity) incubation, the following analysis was conducted: (A) The impact of hMSC (w/o STC1) on the cytotoxicity of CAR-T toward Pfeiffer cells; (B) FACS analysis of CD4+ and CD8+ composition. (C) Quantitation of the FACS data on CD4+ and CD8+; (D) FACS analysis of Treg+ cells (CD4+CD127+CD25+); (E) Quantitation of Treg+ cells. (F) Western blot analysis of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1) expression in the cell co-culture. Data in bar graphs are presented as the mean ± SD from three independent experiments (p values are as indicated, n=3). Figure 2—source data 1 Figure 2A in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data1-v3.xlsx Download elife-82934-fig2-data1-v3.xlsx Figure 2—source data 2 Figure 2C in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data2-v3.xlsx Download elife-82934-fig2-data2-v3.xlsx Figure 2—source data 3 Figure 2E in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data3-v3.xlsx Download elife-82934-fig2-data3-v3.xlsx Figure 2—source data 4 Labeled original blots of Figure 2F. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data4-v3.zip Download elife-82934-fig2-data4-v3.zip Figure 2—source data 5 Unlabeled original blots of Figure 2F. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data5-v3.zip Download elife-82934-fig2-data5-v3.zip Co-culturing with hMSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells Previous studies have demonstrated that the composition of CD4+ and CD8+ T cell subsets was crucial for CAR-T cell efficacy (Sommermeyer et al., 2016; Turtle et al., 2016). To investigate the mechanism of how hMSC inhibited the cytotoxicity of CAR-T, the amount of CD4+ and CD8+ T cells were analyzed by flow cytometry 24 hr after co-culture. As shown in Figure 2B and C, the ratio between CD4+ and CD8+ was about 1:4 when there were no hMSCs in co-culture (Figure 2C). However, the addition of hMSC caused a significant increase of CD4+ and a decrease of CD8+ T cells (Figure 2B), resulting in a ratio change to 2:1. Similar to the change of CD4+ T cells, the percentage of regulatory T cells (Treg) was also significantly increased from ~3 to 12% when co-culture with hMSC (Figure 2D and E). When using hMSCshSTC1, all the changes were completely reversed back to the level similar to that of co-culture without hMSCs. This explains the reduced CAR-T cytotoxicity since CD8+ T cells are directly responsible for specific lytic activity against lymphoma (Sommermeyer et al., 2016). Tregs, which account for 5–10% of the total number of CD4+ T cells, are known to play a role in suppressing the function of T cells and other immune cells (Zhang et al., 2018). Therefore, the above results indicate that hMSCs’ inhibitory effect on CAR-T cytotoxicity was due to both suppression of CD8+ cells and the induction of Treg cells, and the presence of STC1 was indispensable for these impacts of hMSC. The presence of hMSC enhanced immune suppression and STC1 played a key role The immune-suppressive TME is the main cause of CAR-T cell exhaustion which attenuates its efficacy. To further investigate the function of STC1 and the molecular mechanism of hMSC on CAR-T resistance, some key regulators of TME were determined. As shown in Figure 2F, the addition of hMSC to the cell co-culture stimulated the expression of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1). IDO and PD-L1 are two of the most important immunosuppressive proteins. IDO is an intracellular enzyme that converts tryptophan into inhibitory metabolites for T-cell activity (Ninomiya et al., 2015). PD-L1 is expressed in tumor cells and immune cells contributing to the immune-suppressive TME (Ribas and Hu-Lieskovan, 2016). When using hMSCshSTC1, the expression level of IDO and PD-L1 was both significantly reduced by more than 50%, though still higher than that without hMSC. These results indicated that the presence of hMSC can enhance the expression of immune suppressive proteins in Pfeiffer cells and macrophages, and the presence of STC1 is important for hMSC to exert these effects. hMSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial ROS release In the co-culture model, M2 macrophages were included since a previous study showed that macrophages could activate hMSCs to secrete STC1 (Cen et al., 2019). In addition, the macrophage is a critical part of immune response and an important regulator of immunotherapy (DeNardo and Ruffell, 2019). To further identify the mechanisms mediating the inhibitory effects of hMSCs, the activation of the NLRP3 inflammasome was determined. The NLRP3 inflammasome is a critical component of the innate immune system mediating caspase-1 activation and proinflammatory cytokines secretion in response to harmful stimuli such as infection and endogenous stress (Menu and Vince, 2011). As shown in Figure 3A, the release of cleaved caspase-1 p20 in cell lysates, which is the indicator of caspase-1 activation, was detected after the PMA polarization of THP-1 cells to form the M1 macrophages (M-THP1). Following co-culture with CD19 CAR-T, the level of cleaved caspase-1 was significantly upregulated. The increase of active caspase-1 was abrogated when hMSCs were added into the co-culture. knock-down of STC1 led to another reverse and completely blocked the inhibitory function of hMSCs (Figure 3A). Concomitant with the reduction in active caspase-1, the cleaved IL-1β mature form and absent-in-melanoma 2 (AIM2), two key components of the inflammasome (Kelley et al., 2019), were both increasingly expressed following M-THP1 polarization and further incubation with CAR-T (Figure 3A). Compared to the partial inhibition of the active caspase-1 formation, the addition of hMSC in the cell co-culture showed a stronger inhibition of these two proteins, and their expression level was returned to the base level of Pfeiffer plus CAR-T (Figure 3A). This result suggests that the immune-suppressive effect of hMSC was through its impact on macrophages, not CAR-T or Pfeiffer cells. Knockdown of STC1 abrogated the inhibition of hMSC on IL-1β and AIM2 (Figure 3A). The levels of IL-1β in the supernatants by showed similar results as cell (Figure Figure 3 Download asset Open asset The impact of mesenchymal stem cells (MSCs) on the expression of key components involved in the of NLRP3 inflammasome and mitochondrial reactive oxygen species (A) The protein expression of caspase-1, and AIM2 in cell was analyzed by Western (B) Quantitation of IL-1β secretion in the supernatants by (C) FACS analysis of ROS level and with and (D) Quantitation of ROS level based on the percentage of cells that were both for and were 24 hr the co-culture of different cells. For the measurements of results are shown as the mean ± SD from three independent experiments (p values are as indicated, n=3). Figure data 1 Labeled original blots of Figure Download Figure data 2 Unlabeled original blots of Figure Download Figure data 3 Figure in Excel file. Download Figure data 4 Figure in Excel file. Download is one of the stimuli that the NLRP3 and it was reported that STC1 is by macrophages and to to suppress et al., 2009). Therefore, we determined the impact of hMSC on the intracellular level of reactive oxygen species and in macrophages by and As shown in Figure and the presence of suppressed both the cellular and mitochondrial ROS induced by the co-culture of CAR-T cells, tumor cells, and Knockdown of STC1 the function of hMSC in suppressing This result well with the expression of caspase-1, and suggesting that hMSCs inhibited NLRP3 inflammasome activation in macrophages was most by the oxidative hMSCs showed inhibition on CD19 CAR-T therapy in xenograft mice, which was abrogated by STC1 knockdown The immune-suppressive impact of hMSC on CAR-T therapy and the function of STC1 were further evaluated in a xenograft of Pfeiffer cells and of we hMSC into the tumor CAR-T treatment by As shown in Figure CD19 CAR-T treatment with the of a significant effect, and the tumors at However, the showed a increase in tumor and of Figure 4 Download asset Open asset The inhibition of hMSC on chimeric antigen receptor modified T cell (CAR-T) therapy in xenograft mice on (A) The and progression of tumor in three of mice with the control without any treatment, and was when the was confirmed after the Pfeiffer cells. (B) analysis of and Treg cells as the in tumor tissue at cells or (C) The tumor change with (D) The presented as the mean ± SD (p values are as indicated, n=3). Figure data 1 Figure in Excel file. Download Figure data 2 Figure in Excel file. Download Based on the analysis of IL-1β in tumor tissue on the number of cells from to in the it from 5 to in the that hMSC could suppress TME and STC1 knockdown significantly this impact (Figure with the results in a large amount of CD4+ T cells were detected in the but much less in the On the the amount of CD8+ T cells was significantly increased in the compared to that of the (Figure Based on the of a master regulator involved in the development of Treg cells, the amount of Treg cells was also increased in the compared to that of the (Figure These results further confirmed that knockdown of STC1 abrogated the immune-suppressive of MSC. The changes in the were with the changes in the tumor (Figure and The survival time of mice demonstrated that mice in CAR-T with the had the survival with no by (Figure Compared to the control with no CAR-T treatment, tumor in the was and all for These results confirmed the inhibitory effects of hMSC on CAR-T therapy in and demonstrated that STC1 is an important factor therapy efficacy. Discussion Stem cells are believed to play critical roles in resistance to cancer therapy, which is a to poor treatment responses and tumor Previous studies have been on the role of cancer stem cells. In the we presented that the presence of MSCs in TME may also be an important of cancer treatment resistance. By modulating MSCs showed a suppressive function on CAR-T efficacy toward lymphoma cells, and the presence of the STC1 gene played a critical role. The role of STC1 in cancer is Some reports showed that it an oncogenic whereas other studies the et al., 2019). The expression of STC1 has been reported to impact various types of such as tumor by the expression of in cancer cells et al., and poor clinical in and cancers (Yeung et al., 2012; et al., 2019). To the potential roles of STC1 in immunotherapy are still we demonstrated that the presence of STC1 is critical for MSC to exert its immunosuppressive role by T cell some key immune and with other immune cells. a significant of CD8+ T together with the of CD4+ T cell subsets and indicated that the suppressed CAR-T efficacy was at partially associated with function in modulating the proliferation of different T-cell the suppression of CD8+ T cells was completely abrogated if knockdown STC1 in MSCs, it is that STC1 played a key role Moreover, that STC1 is into the extracellular in a paracrine of the T cell subsets is most cytokine expression or other molecules activated by STC1. In line with our it was reported that STC-1 with immunotherapy and T cell activation by which membrane antigen function and et al., 2021). The presence of MSCs also stimulated the expression of IDO and two important immune-suppressive of IDO is an endogenous mechanism controlling excessive immune which can be produced both by tumor cells and macrophages et al., of immunosuppressive metabolites can T-cell proliferation and T-cell through the receptor et al., 2011; et al., PD-L1 is a of the mechanism of immunotherapy by T cell function and antigen (Ribas and Hu-Lieskovan, 2016). There have been studies the between MSCs and cancer cells, resulting in regulating the expression of PD-L1 on the of various cancer cells or TME and et al., 2019; et al., et al., 2018). here we demonstrated that the expression of both IDO and PD-L1 by MSCs was much reduced if the STC1 gene was

It is well established that multifactorial drug resistance hinders successful cancer treatment. Tumor cell interactions with the tumor microenvironment (TME) are crucial in epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR). TME-induced factors secreted by cancer cells and cancer-associated fibroblasts (CAFs) create an inflammatory microenvironment by recruiting immune cells. CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) and inflammatory tumor associated macrophages (TAMs) are main immune cell types which further enhance chronic inflammation. Chronic inflammation nurtures tumor-initiating/cancer stem-like cells (CSCs), induces both EMT and MDR leading to tumor relapses. Pro-thrombotic microenvironment created by inflammatory cytokines and chemokines from TAMs, MDSCs and CAFs is also involved in EMT and MDR. MDSCs are the most common mediators of immunosuppression and are also involved in resistance to targeted therapies, e.g. BRAF inhibitors and oncolytic viruses-based therapies. Expansion of both cancer and stroma cells causes hypoxia by hypoxia-inducible transcription factors (e.g. HIF-1α) resulting in drug resistance. TME factors induce the expression of transcriptional EMT factors, MDR and metabolic adaptation of cancer cells. Promoters of several ATP-binding cassette (ABC) transporter genes contain binding sites for canonical EMT transcription factors, e.g. ZEB, TWIST and SNAIL. Changes in glycolysis, oxidative phosphorylation and autophagy during EMT also promote MDR. Conclusively, EMT signaling simultaneously increases MDR. Owing to the multifactorial nature of MDR, targeting one mechanism seems to be non-sufficient to overcome resistance. Targeting inflammatory processes by immune modulatory compounds such as mTOR inhibitors, demethylating agents, low-dosed histone deacetylase inhibitors may decrease MDR. Targeting EMT and metabolic adaptation by small molecular inhibitors might also reverse MDR. In this review, we summarize evidence for TME components as causative factors of EMT and anticancer drug resistance.

Soiling the Seed: Microenvironment and Epithelial Mesenchymal Plasticity

Cancer Cell Metabolism: Warburg and Beyond

P3.03-11 Lung Cancer Stem Cells on Immune Modulation in Tumorous Microenvironment

Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy. Citation Format: Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4089.

The tumor microenvironment (TME) can be defined as non-transformed stromal elements within the tumor, effecting their growth, invasion, and metastatic ability. Cancer cells leave the primary tumor during metastasis, then spread and colonize in distant organs. In breast cancer, metastasis mechanisms are not yet fully understood within the TME. However, epithelial to mesenchymal transition (EMT) is a known essential step in metastasis, in which patterned epithelial cells transform into migratory cells with mesenchymal phenotypes. Developing state-of-the-art multiparametric in vivo imaging devices increases the opportunities to observe dynamic cellular properties and their interactions with the surrounding stroma and TME during the critical steps of invasion and metastasis. Currently available commercial imaging technologies only enable in vivo imaging under anesthesia in a fixed posture. In contrast, real-time confocal imaging of the EMT and TME is not possible in animals. We have adapted our miniature dual-axis confocal (DAC) fluorescent microscope to track breast cancer cells having fluorescent reporters indicating key molecular splicing events during EMT to overcome these limitations. This project aims to study EMT more thoroughly using rat breast cancer MTLn3 cell lines by mimicking in vivo scenarios through 3D in vitro mono/co-culture spheroids, 3D multi-spheroid matrix overlay models, and 3D sandwich. We have engineered MTLn3 cells with fluorescent reporters correlating with different EMT stages, depending on their invasiveness levels. The ultra-low attachment method is used to engineer 3D MTLn3 spheroids, embed within TME matrices, and monitor advanced in vitro live-cell imaging techniques for up to 10 days. Another goal is to use miniature DAC microscopes, implanted in animal models, to study the fundamental changes occurring during carcinoma progression. This transdisciplinary project's final goal is to optimize the DAC microscope as an implantable and insertable device to investigate EMT-associated splicing events in the TME of orthotopic rat breast carcinoma models. This will be achieved by growing engineered MTLn3 cells and spheroids in their mammary fat pads and following microscopic changes for a certain period. This work will contribute to further developing the miniature implantable DAC microscopes for 3D high resolution, multiparametric imaging to reveal dynamic changes within the TME during carcinoma progression. Citation Format: Ehsanul Hoque Apu, Seock-Jin Chung, Michael J. Mandella, Frank Schonig, Frank B. Gertler, Zhen Qiu, Christopher H. Contag. Implantable and insertable miniature dual-axis confocal (DAC) microscope to detect epithelial-mesenchymal transition (EMT) in the breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB243.

Immunotherapy Exposes Cancer Stem Cell Resistance and a New Synthetic Lethality

Epithelial–Mesenchymal Transition in Tumor Metastasis: A Method to the Madness

Pan-cancer analysis of altered glycosyltransferases confers poor clinical outcomes.

Pancreatic cancer remains one of the most deadly cancers, with over 450,000 fatalities in 2022 alone. The most lethal variation of this cancer is Pancreatic Ductal Adenocarcinoma (PDAC), with a 5-year survival rate of under 10%. Combination chemotherapy has provided major advances in this disease but it remains challenging for many reasons including the immunosuppressive nature of the tumor microenvironment (TME). PDAC has a highly desmoplastic microenvironment that suppresses antitumor immunity and promotes tumor cell invasion, metastasis, and resistance through epithelial to mesenchymal transition (EMT) of cancer cells. Cancer cells can undergo various forms of cell death, notably apoptosis, necrosis, autophagy, pyroptosis, and ferroptosis. These mechanisms are triggered by different cellular stressors and shape the microenvironment. Through the use of 15 Visium spatial transcriptomics samples from publicly available primary PDAC tumors and paired single-cell RNA (scRNA) data, we aim to understand how cell stress and cell death shape the tumor microenvironment (TME) and epithelial-mesenchymal transition (EMT). By leveraging information from known pathways in literature, we will identify cellular programs related to stress, death, and EMT. We will explore intracellular correlations within single-cell data and extracellular interactions within the TME using spatial data to uncover how different forms of cell death and stress signatures influence the TME and EMT spectrum. The initial phase involves identifying the types of stress that lead to various forms of cell death intracellularly and validating these gene signatures. Subsequently, we will apply these validated signatures to the spatial data to correlate each score and search for significant overlapping programs. This will extend beyond stress and death, incorporating TME signatures corresponding to different cell types and the EMT spectrum. By understanding the signals that promote the death of various cell populations, we aim to identify combination treatments that target both ends of the EMT spectrum. Additionally, this research will provide insights into the inflammatory impact of cell death on the TME. Our approach aims to inform the development of therapies that disrupt resistance mechanisms, ultimately improving treatment outcomes for patients with PDAC. This research can potentially reveal new therapeutic targets and strategies, enhancing our ability to combat this deadly disease. Citation Format: Rahul Bansal, Izabella Zamora, Samuel Wright, Nir Hacohen, Arnav Mehta. Influence of Cell Death and Stress on the Tumor Microenvironment and EMT in Pancreatic Ductal Adenocarcinoma Through Single Cell and Spatial Transcriptomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C074.

The tumor microenvironment influences cancer cell behavior in relation to tumor progression, as well as cell proliferation and invasion. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic reaction and extracellular matrix (ECM) components in the tumor microenvironment are involved in a cross-talk between tumor cells, stromal fibroblasts and ECM components, influencing tumor cell behavior. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and the ECM of the microenvironment by culturing PDAC cells on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell phenotype and the proteins occurring in the desmoplastic tissue. For this purpose, we analyzed some epithelial-to-mesenchymal transition (EMT) markers and the migration and invasive potential in human HPAF-II, HPAC and PL45 PDAC cells cultured on collagen type I (COL), laminin (LAM) and fibronectin (FN). Interestingly, the expression of E-cadherin was not significantly affected, but some differences were revealed by the wound healing assay. In fact, migration of HPAF-II and PL45 cells was decreased on FN and LAM, and increased on COL, compared to control cells grown on plastic (NC). By contrast, HPAC was very rapid and unaffected by the substrate. SDS-zymography showed that COL induced a strong upregulation of MMP-2 activity in HPAF-II and HPAC cells, and of MMP-9 in HPAF-II and PL45 cells, compared to NC. These preliminary results suggest that ECM components could differently affect PDAC migration and invasion, possibly depending on the differentiation grade. The characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies.