Stromal Markers Research Articles

Abstract 3124: An emerging paradigm of Cxcl12/Cxcr4 involvement in breast cancer metastasis

Abstract The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma and to be associated with increased metastatic risk in humans. Indeed, prior studies have specifically linked Cxcl12/Cxcr4 to breast cancer cell seeding, homing, survival and proliferation at future metastatic sites, due to the aberrant Cxcl12 expression in these sites (e.g. lung, liver and bone marrow). Interestingly however, the precise mechanism via which Cxcr4+ breast cancer cells escape the primary tumors in the first place (which also highly express Cxcl12), remains poorly understood. By using a novel methodology for quantifying chemotactic gradients using fixed tissue multichannel immunofluorescence (mIF), here, we demonstrate in mouse primary breast tumors that Cxcl12 gradients are concentrically expressed around cancer cell intravasation sites, known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms using mIF, we also demonstrate that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM-mediated metastatic dissemination. However, targeted elimination of the Cxcr4+ gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells utilizing TMEM doorways. We thus theorized that, in the absence of Cxcr4 expression in tumor cells, the accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM-generated Cxcl12 gradients in mouse primary breast cancers. Despite that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm for the implication of the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and propose a new avenue for rationalized antimetastatic treatments for breast cancer. Citation Format: Maria K. Lagou, Luis G. Rivera, Camille E. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12/Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3124.

Abstract 1579: Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression

Abstract Background. Stromal fibroblasts, the predominant cell type within the tumor microenvironment, plays a critical regulatory role in prostate cancer (PC) by engaging in stromal-epithelial interactions to dictate cancer initiation, growth, and progression. Monoamine oxidase B (MAOB) catalyzes oxidative deamination of biogenic and dietary monoamines and generates hydrogen peroxide as a by-product, which can predispose stromal cells to a reactive, cancer-promoting state with elevated production of extracellular matrix molecules, growth factors, cytokines, and chemokines. This study investigated the clinical relevance and previously unrecognized function of stromal MAOB in PC. Methods. The stromal MAOB expression levels were determined in PC clinical specimens by immunohistochemistry and in publicly available multi-omics cancer datasets, which were associated with different clinical parameters. The human prostatic stromal fibroblast PrSC cells engineered for stable overexpression or knockdown of MAOB were co-cultured with a panel of human PC cells in 2-D or 3-D fashion, followed by examination of cancer cell behavior by cell proliferation, migration, and invasion assays. MAOB-knockdown PrSC cells were co-inoculated with luciferase-tagged PC cells as subrenal tissue recombinant xenografts into mice for assessment of MAOB’s impact on tumor growth by bioluminescence imaging. Results. MAOB expression was induced in the stroma associated with primary PC compared to normal prostatic tissues of clinical samples, which was corroborated by the same MAOB expression trend in three matched pairs of patient-derived fibroblasts. Following the analysis of three independent clinical cohorts, increased stromal MAOB levels were shown to be correlated with higher Gleason scores, castration resistance, survival, and appearance of neuroendocrine differentiation as evidenced by CHGA expression in the adjacent epithelia. Co-culturing MAOB-overexpressing PrSC cells with several human PC cell lines (C4-2, PC-3, DU145 and C4-2BENZR) accelerated cancer cell proliferation, migration, and invasion. Conversely, these cancer cell phenotypes were suppressed in the presence of MAOB-knockdown PrSC cells. Additionally, C4-2 tumor xenografts when co-inoculated with MAOB-knockdown PrSC grew to a substantially smaller size in mice, which was paralleled by reduced Ki-67 index and reactive stromal marker αSMA expression in the tumor and stromal compartments respectively, compared to controls. Conclusion. Our results suggest that the elevated MAOB expression in PC stromal cells is associated with poor clinical outcomes and further contributes to adjacent PC growth, which provides new insights into understanding the stromal support of PC pathogenesis and progression. Funding provided by the NIH/NCI grant R01CA258634 and WSU CPPS startup fund (to B.W). Citation Format: Tainjie Pu, Jing Wang, Chia-Hui Chen, Tzu-Ping Lin, Boyang Wu. Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1579.

Thrombospondin-2 acts as a bridge between tumor extracellular matrix and immune infiltration in pancreatic and stomach adenocarcinomas: an integrative pan-cancer analysis

BackgroundThrombospondin-2 (THBS2) is a versatile glycoprotein that regulates numerous biological functions, including the apoptosis-proliferation balance in endothelial cells, and it has been linked to tumor angiogenesis. However, the exact role of THBS2 in human cancer remains unknown. This study aimed to determine THBS2 expression in a pan-cancer analysis and its association with pan-cancer prognosis and to further identify its possible roles in tumor immunity and the extracellular matrix (ECM).MethodsData on THBS2 expression in cancers and normal tissues were downloaded from the Genotype-Tissue Expression portal and UCSC Xena visual exploration tool and analyzed using the ONCOMINE database, Perl programming language, and Gene Expression Profiling and Interactive Analyses vision 2 webserver. In addition, survival prognosis was analyzed using the survival, survminer, limma, and forestplot packages in R v. 4.0.3.Immune and matrix components were also analyzed using R v. 4.0.3. Most importantly, we partially validated the role and mechanism of THBS2 in pancreatic and gastric cancers in vitro using PANC1 and BGC-823 cell lines.ResultsTHBS2 was significantly overexpressed in 17 of the 33 investigated cancers and linked to a poor prognosis in pan-cancer survival analysis. High THBS2 expression was an independent unfavorable prognostic factor in kidney renal papillary cell, mesothelioma, and stomach and pancreatic adenocarcinomas. Immune infiltration and THBS2 expression were also related. THBS2 expression has been linked to immune and stromal scores and immune checkpoint markers in various cancers. The protein–protein interaction network revealed that THBS2 is associated with multiple ECM and immune proteins. THBS2 knockdown decreased the expression of CD47 and matrix metallopeptidase 2 (MMP-2) as well as the proliferation, migration, and invasion of PANC1 and BGC-823 cells in vitro.ConclusionsOur findings suggested that THBS2 might promote cancer progression by remodeling the tumor microenvironment, affecting CD47-mediated signaling pathways, activating the pro-tumor functions of a disintegrin and metalloproteinase with thrombospondin motifs, and enhancing MMP-2 expression. Furthermore, it functions as a bridge between the ECM and immune infiltration in cancer and serves as a potential prognostic biomarker for several cancers, especially pancreatic and gastric adenocarcinomas.

Anti-PD1, capecitabine, and oxaliplatin for the first-line treatment of dMMR esophagogastric cancer (AuspiCiOus-dMMR): A proof-of-principle study (AuspiCiOus).

TPS4163 Background: Doublet cytotoxic treatment demonstrates survival benefit over single drug therapies in patients with advanced esophagogastric cancer (EGC). Previous clinical data suggests an improved survival outcome in patients with microsatellite instability (MSI) treated with PD-1 inhibitor monotherapy or in combination with chemotherapy in first line palliative treatment. The positive effect of immunotherapy was only seen after a couple of months of treatment. This suggests that the patient might benefit from a short course of chemotherapy at the start of treatment. However, reports on the effects of cytotoxic treatment in MSI-high tumors range from beneficial to detrimental. Thus, the value of cytotoxic treatment, as well as the effect on the tumor immune microenvironment (TME) in MSI-high tumors is unclear. Therefore, in AuspiCious-dMMR we will study the effect of sequential treatment in patients with mismatch repair deficient (dMMR) EGC on the tumor immune microenvironment and, more specifically, the impact on the infiltration of cytotoxic T cells, before and after a short course of chemotherapy, and during treatment with PD-1 inhibition. Methods: AuspiCiOus is a multi-center, open-label, single arm (phase 2) study in patients with dMMR irresectable or metastatic EGC adenocarcinoma (NCT05177133). Patients are treated with Capecitabine and Oxaliplatin for two 3-weekly cycles, after which treatment is continued with retifanlimab (PD-1 inhibitor) for 4-weekly cycles up to progression or unacceptable toxicity, with a maximum of two years. Before treatment, after chemotherapy and after two cycles of immunotherapy fresh tumor biopsies are collected to study the primary outcome (T cell infiltration and the interferon-γ signature) as well as for translational research purposes (flow cytometry, nanostring profiling, (multicolor) immunohistochemistry and organoid culturing). Blood is collected at the same time points and on day 3 after the first course of immunotherapy for isolation of peripheral blood mononuclear cell, and determination of cytokine/chemokine profile, circulating stromal markers, and circulating tumor DNA. Also, fecal samples are collected to determine changes in the intestinal microbiome and patient reported outcome measures are used to determine quality of life. (s)AEs are assessed according to CTCAE v5 and tumor response is determined according to (i)RECIST 1.1. Key eligibility criteria are 1) histological confirmed diagnosis of untreated metastatic or irresectable HER2- adenocarcinoma of stomach or junction and 2) dMMR determination by IHC. Study is open for inclusion and three of planned 25 patients have been enrolled. Clinical trial information: NCT05177133.

Candidate diagnostic and prognostic markers for pancreatitis and pancreatic ductal adenocarcinoma

Diagnostic markers are desperately needed for early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity of PDA marker expression was analyzed by interrogating quantitative dot plots of single cell (sc) RNAseq for 3021 candidate genes in tumor samples from a mouse model for PDA (KIC) at early and late stages of PDA progression compared to normal pancreas. Protein markers were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines. Additional candidate genes were identified from signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes. Developmental progression of cancer was revealed in seven classes of PDA markers expressed in cancer cells and stroma. The earliest diagnostic markers (47 genes) were specifically expressed in epithelial cancer cells in early, but not late, PDA tumors. Other markers were first elevated in epithelial cancer cells of late stage tumors (288 genes), or in both epithelial and mesenchymal cells (740 genes), or only in mesenchymal cells (216 genes). Stromal markers were differentially expressed in early‐ and/or late‐PDA neoplasia (594 genes). Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and acinar‐specific homozygous deletion of differentiation transcription factors (dTFs), were screened for early expression of all PDA markers identified in KIC neoplasia. Select diagnostic markers were validated by immunohistochemistry in mouse and human samples of normal pancreas, early‑ and late‐stage PDA. Prognostic markers from TCGA were identified that showed differential and cell type‐specific expression in PDA. Here we present diagnostic and prognostic markers for disease progression from pancreatitis to late‐stage PDA.

Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles.

Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer‐associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell–cell communication. Epithelial CRC EVs suppressed TGF‐β‐driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR‐200 (miR‐200a/b/c, ‐141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR‐200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR‐200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co‐injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR‐200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV‐mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich.

Serum-based measurements of stromal activation through ADAM12 associate with poor prognosis in colorectal cancer

BackgroundRecently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC).MethodsUsing gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20).ResultsADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11–1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06–3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25–3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers.ConclusionsHere we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum.

Arsenic exposure impairs intestinal stromal cells

Arsenic is a toxicant commonly found in drinking water. Even though its main route of exposure is oral, little is known of the impact of in vivo arsenic exposure on small intestine. In vitro studies have shown that arsenic decreases differentiation of stem and progenitor cells in several different tissues. Thus, small intestinal organoids were used to assess if arsenic exposure would also impair intestinal stem cell differentiation. Unexpectedly, no changes in markers of differentiated epithelial cells were seen. However, exposing mice to 100 ppb arsenic in drinking water for 5 weeks impaired distinct populations of intestinal stromal cells. Arsenic reduced the width of the pericryptal lamina propria by 1.6-fold, and reduced Pdgfra mRNA expression, which is expressed in intestinal telocytes and trophocytes, by 4.2-fold. The height or extension of Pdgfra+ telopodes into the villus tip was also significantly reduced. Transcript expression of several other stromal cell markers, such as Grem1, Gli, CD81, were reduced by 1.9-, 2.3-, and 1.4-fold, respectively. Further, significant correlations exist between levels of Pdgfra and Gli1, Grem1, and Bmp4. Our results suggest arsenic impairs intestinal trophocytes and telocytes, leading to alterations in the Bmp signaling pathway.

Myofibroblasts as important diagnostic and prognostic indicators of oral squamous cell carcinoma: An immunohistochemical study using alpha-smooth muscle actin antibody.

Background:Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity, with multifactorial etiopathogenesis. Data from the past literature suggest that myofibroblasts (MFs) can also contribute significantly to the pathogenesis of the disease. Hence, the present study was undertaken for assessing the expression of MF in well-differentiated OSCC (WDOSCC), moderately differentiated OSCC (MDOSCC), poorly differentiated OSCC (PDOSCC) and healthy controls by immunohistochemistry using alpha-smooth muscle actin (α-SMA) antibody.Methodology:Forty cases each of WDOSCC, MDOSCC, PDOSCC and healthy controls were included. 4-μm thick sections from each tissue sample were stained with routine hematoxylin and eosin as well as immunohistochemically using α-SMA. Among different grades of OSCC, expression of MFs was compared. All the results were subjected to statistical analysis.Results:While comparing the expression of MFs in between different grades of OSCC, nonsignificant results were obtained. While comparing the expression of MF in between OSCC cases and normal controls, significant results were obtained.Conclusion:MFs are one of the vital pathogenetic components in OSCC cases in predicting their invasive behaviors. We advocate the use of MFs as a stromal marker for visualizing invasion and progression in OSCC patients.

Pathological response and predictive role of tumour-infiltrating lymphocytes in HER2-positive early breast cancer treated with neoadjuvant pyrotinib plus trastuzumab and chemotherapy (Panphila): a multicentre phase 2 trial

PurposePanphila evaluated pyrotinib plus trastuzumab, docetaxel and carboplatin as neoadjuvant therapy for early breast cancer (BC), and investigated the predictive role of immune cell subpopulations. Patients and methodsIn this multicentre phase 2 study, patients with human epidermal growth factor receptor 2-positive, stage T2-3N0-3M0 BC received pyrotinib 400 mg once daily plus docetaxel (75 mg/m2, day 1), carboplatin (6 mg/mL/min, day 1) and trastuzumab (8 mg/kg loading dose and 6 mg/kg maintenance dose, day 1) for 6 cycles of 21 days each. Simon's 2-stage design was adopted. The primary end-point was pathological complete response (pCR, ypT0/is ypN0) rate. Tumour-infiltrating lymphocytes (TILs) were assessed by haematoxylin and eosin staining and multiplex immunohistochemistry. ResultsIn the modified intention-to-treat population (n = 69), 38 patients (55.1%) achieved pCR. In the safety population (n = 74), the most common grade ≥3 adverse events were diarrhoea (43.2%), anaemia (37.8%), vomiting (16.2%) and platelet count decrease (10.8%). No treatment-related deaths occurred. Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by stromal (s)-CD20+, s-CD8+ and s-CD4+ TILs. Unsupervised hierarchical clustering of stromal immune markers identified a group of patients characterised by high s-CD20+, s-CD8+, s-CD4+ and s-FOXP3+ immune cells infiltration, which was independently associated with pCR. ConclusionNeoadjuvant pyrotinib plus trastuzumab-based chemotherapy exhibits promising efficacy and manageable toxicity in patients with human epidermal growth factor receptor 2-positive early BC, and thus phase 3 trials are warranted. Our findings also contribute to understanding the potential role of the immune microenvironment in response to neoadjuvant pyrotinib-based therapy.

Insulin facilitates corneal wound healing in the diabetic environment through the RTK-PI3K/Akt/mTOR axis in vitro

Diabetic patients can develop degenerative corneal changes, termed diabetic keratopathy, during the course of their disease. Topical insulin has been shown to reduce corneal wound area and restore sensitivity in diabetic rats, and both the insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) stimulate cell signaling of the PI3K-Akt pathway. The purpose of this study was to assess a mechanism by which improved wound healing occurs by characterizing expression within the PI3K-Akt pathway in corneal epithelial and stromal cells. In vitro scratch tests were used to evaluate wound healing outcomes under variable glucose conditions in the presence or absence of insulin. Protein expression of intracellular kinases in the PI3K pathway, stromal cell markers, and GLUT-1 was evaluated by immunoblotting.TGF-β1 expression was evaluated by ELISA. Insulin promoted in vitro wound healing in all cell types. In human corneal epithelial cells, insulin did not induce PI3K-Akt signaling; however, in all other cell types evaluated, insulin increased expression of PI3K-Akt signaling proteins compared to vehicle control. Fibroblasts variably expressed α-SMA under all treatment conditions, with significant increases in α-SMA and TGF-β1 occurring in a dose-dependent manner with glucose concentration. These results indicate that insulin can promote corneal cellular migration and proliferation by inducing Akt signaling. Exogenous insulin therapy may serve as a novel target of therapeutic intervention for diabetic keratopathy.

A Novel Superpixel Approach to the Tumoral Microenvironment in Colorectal Cancer

Colorectal cancer (CRC) is the most common malignancy of the gastrointestinal tract. The stroma and the tumoral microenvironment (TME) represent ecosystem-like biological networks and are new frontiers in CRC. The present study demonstrates the use of a novel machine learning-based superpixel approach for whole slide images to unravel this biology. Findings of significance include the association of low proportionated stromal area, high immature stromal percentage, and high myxoid stromal ratio (MSR) with worse prognostic outcomes in CRC. Overall, stromal computational markers outperformed all others at predicting clinical outcomes. MSR may be able to prognosticate patients independent of pathological stage, representing an optimal way to effectively prognosticate CRC patients which circumvents the need for more extensive molecular and/or computational profiling. The superpixel approaches to the TME demonstrated here can be performed by a trained pathologist and recorded during synoptic cancer reporting with appropriate quality assurance. Future clinical trials will have the ultimate say in determining whether we can better tailor the need for adjuvant therapy in patients with CRC.

A Quick and Efficient Method for the Generation of Immunomodulatory Mesenchymal Stromal Cell from Human Induced Pluripotent Stem Cell.

Mesenchymal stromal cells (MSCs) have been widely investigated for their regenerative capacity, anti-inflammatory properties and beneficial immunomodulatory effects across multiple clinical indications. Nevertheless, their widespread clinical utilization is limited by the variability in MSC quality, impacted by donor age, metabolism, and disease. Human induced pluripotent stem cells (hiPSCs) generated from readily accessible donor tissues, are a promising source of stable and rejuvenated MSC but differentiation methods generally require prolonged culture and result in low frequencies of stable MSCs. To overcome this limitation, we have optimized a quick and efficient method for hiPSC differentiation into footprint-free MSCs (human induced MSCs [hiMSCs]) in this study. This method capitalizes on the synergistic action of growth factors Wnt3a and Activin A with bone morphogenetic protein-4 (BMP4), leading to an enrichment of MSC after only 4 days of treatment. These hiMSCs demonstrate a significant upregulation of mesenchymal stromal markers (CD105+, CD90+, CD73, and cadherin 11) compared with bone marrow-derived MSCs (bmMSCs), with reduced expression of the pluripotency genes (octamer-binding transcription factor [Oct-4], cellular myelocytomatosis oncogene [c-Myc], Klf4, and Nanog homebox [Nanog]) compared with hiPSC. Moreover, they show improved proliferation capacity in culture without inducing any teratoma formation in vivo. Osteogenesis, chondrogenesis, and adipogenesis assays confirmed the ability of hiMSCs to differentiate into the three different lineages. Secretome analyses showed cytokine profiles compared with bmMSCs. Encapsulated hiMSCs in alginate beads cocultured with osteoarthritic (OA) cartilage explants showed robust immunomodulation, with stimulation of cell growth and proteoglycan production in OA cartilage. Our quick and efficient protocol for derivation of hiMSC from hiPSC, and their encapsulation in microbeads, therefore, presents a reliable and reproducible method to boost the clinical applications of MSCs.

Decidualization Potency and Epigenetic Changes in Human Endometrial Origin Stem Cells During Propagation.

Human endometrium derived mesenchymal stem cells (hEndSCs) offer a great promise for regenerative medicine and reproductive system disorders treatment methods based on cell therapy due to their broad differentiation potential and highly efficient proliferation. In our study, we investigated the characteristics of hEndSCs that were isolated from two sources: endometrium and menstrual blood, which both contain endometrial origin stem cells. Changes in gene and protein expression levels during long-term cultivation and decidualization potential were examined in endometrial stem cells (EndSCs) and menstrual blood stem cells (MenSCs). The decidualization process was induced on early and late passages of hEndSCs using dibutyryl cyclic-AMP (db-cAMP) and medroxyprogesterone acetate (MPA) agents. We demonstrated that after long-term cultivation of hEndSCs the expression of typical mesenchymal stromal cell surface markers such as CD44, CD73, CD90, CD105 and perivascular marker CD146 remains at a similar level throughout long-term cultivation. Additionally, hematopoietic and endothelial markers CD34, CD45 were also tested, they were negative in all cases. Analyzed stem cells gene markers, such as OCT4, SOX2, NANOG, KLF4, showed similar expression in all passages of hEndSCs. RT-qPCR results demonstrated that the expression of cell cycle control associated genes - CDK2, CCNA2, CCNE2, p21, p53 and Rb, among all groups was very similar. Expression of genes associated with senescence (ATM, JUND, TOP2A, MYC) was maintained at a similar level throughout passaging. In addition, Western blot analysis was used to assess changes in proteins’ levels associated to epigenetics (EZH2, SUZ12, H3K27me3) and cell cycle control (cyclinE1, p53) during long-term cultivation. The levels of proteins associated with epigenetic changes were fluctuated slightly depending on the patient. Also, we demonstrated that in all induced hEndSCs the expression of decidualization markers Prolactin (PRL), IGFBP1 and WNT4 was upregulated. In conclusion, we demonstrated successful decidualization of stem cells derived from two reproductive system resources: endometrium and menstrual blood by using db-cAMP and MPA regardless of the length of the stem cell passaging. According these findings, we suppose that endometrium derived stem cells and menstrual blood derived stem cells could have a potency not only for endometrium tissue regeneration, but could also become a successful therapy for reproductive system disorders, including infertility or recurrent pregnancy loss.

Abstract PO-117: The role of Hippo signaling in stromal-epithelial interactions in acinar-to-ductal metaplasia and pancreatic cancer initiation

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with a 5-year survival rate of approximately 7%. PDAC may originate from acinar cell trans-differentiation into ductal-like cells, termed acinar-to-ductal metaplasia (ADM), triggered by chronic pancreatitis and/or mutations in K-Ras. The progression to PDAC is associated with a dense fibrotic stroma, including cancer-associated fibroblasts (CAFs). YAP is a tension-stimulated CAF activator that promotes ECM stiffening, creating a permissive microenvironment for cancer progression. We hypothesize that the Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury and progression towards PDAC. Methods: To resolve the transcriptional changes occurring during the transition to ADM and PDAC, we mapped the in situ expression of over 1800 RNA targets in patient-derived tissues using NanoString Technologies’ Digital Spatial Profiling (DSP) technology. We also performed immune-profiling and evaluated Yap expression in human ADM by immunohistochemistry. To study the in vivo role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis, which recapitulates many of the features associated with human ADM. I will analyze the resulting phenotype by immunostaining for metaplastic, proliferative, immune and stromal markers. Results: DSP analysis revealed genes implicated in fibroblast activation, epithelial-to-mesenchymal transition (EMT), neutrophil activation and IFNγ signaling as potential key drivers of ADM. I will further evaluate the expression of candidate genes and survey Yap expression at the single cell level in human ADM tissue by multiplexed RNAscope in situ hybridization. We found up-regulation of CD4+ and CD8+ T cells in ADM, and an increasing trend of neutrophil and macrophage accumulation in the progression from normal parenchyma to ADM to PDAC. Conclusions: This work will provide an in-depth understanding of epithelial-stroma crosstalk in ADM and a foundation for the development of new therapeutic strategies for treating non-invasive precursor lesions like ADM, thereby preventing pancreatic cancer progression. Source of Funding: This research is supported by the Fonds de Recherce du Quebec – Santé (FRQS), Canadian Institutes of Health Research (CIHR) and the Research Institute of the McGill University Health Centre (RI-MUHC). Citation Format: Julia Messina-Pacheco, Yasser Riazalhosseini, Zu-hua Gao, Alex Gregorieff. The role of Hippo signaling in stromal-epithelial interactions in acinar-to-ductal metaplasia and pancreatic cancer initiation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-117.

Dynamic Stromal Changes in Myelofibrosis Patients Pre/Post JAK Inhibition Is Revealed in Clinically Archived Bone Marrow Biopsies By Smooth Muscle Actin (SMA)-CD34 Dual Immunohistochemistry

The natural history of BCR-ABL1 negative myeloproliferative neoplasms (MPNs) is progression towards an overt myelofibrotic (MF) phase with variable risk to develop secondary acute myeloid leukemia. Current treatments include Janus kinase inhibitors (JAKi) which can temporarily alleviate MF-related symptoms but are non-curative and most patients eventually progress to a more advanced stage. Given the negative prognostic impact of bone marrow fibrosis in MPNs and generally poor outcome post JAKi failure, it would be important to identify in situ biomarkers that address the initiation, perpetuation and early reversal of the fibrotic reaction.The current clinical standard for bone marrow fibrosis assessment involves reticulin/trichrome stains that detect relatively static extracellular matrix products rather than the fibrosis driving cells directly. To address this, we have developed a smooth muscle actin stromal-vascular (SMA-CD34) dual immunohistochemical (IHC) technique amenable to morphologic scoring and complemented with a CellProfiler image analysis pipeline. SMA was prioritized over other validated stromal IHC markers given work by others in experimental models demonstrating SMA+ myofibroblasts to be the differentiated output of critical fibrosis inducing Gli1+ ‘driver’ mesenchymal stem/progenitor cells in MPN.Herein, we demonstrate the feasibility of our translational approach using a clinically annotated cohort of MF patients from the Princess Margaret Cancer Centre MPN Registry. After selecting for high quality (>1.0 cm) paired pre and post JAKi biopsies amenable to image and transcriptome-based analysis, the pilot cohort was comprised of 13 cases with 38% high-risk, 54% intermediate-2 and 8% intermediate-1 risk by DIPSS. Driver mutations were JAK2 V617F (77%), CALR (15%) and other (8%). JAKi therapies included ruxolitinib (31%) + pelabresib (23%), momelotinib (15%), itacitinib (15%) and pacritinib (8%).The SMA-CD34 stromal assessment at baseline revealed distinct interstitial myofibroblast patterns and vascular perturbations not captured by conventional clinical hematopathology assessment (e.g. SMA+ dilated sinusoids). A SMA-CD34 scoring system was developed using a 4-point scale representing normal (0 pts), increased vascularity (1 pt), focal interstitial SMA (2 pts), multifocal interstitial SMA (3 pts) and diffuse SMA (4 pts). Scoring was then performed by blinded hematopathologists. A trend towards JAK2 mutated MF cases demonstrating higher SMA grade at baseline was noted. Interestingly, variable trajectories in SMA scores emerged following treatment with JAKi. Specifically, SMA signals had increased in 15%, decreased in 46% and were stable in 38% post-JAKi when using a morphologic SMA grading scheme. When compared to reticulin fibrosis, the severity of SMA signals had diverged in 1/3 of the cases (e.g. SMA grade decreased, reticulin grade stable).To further complement the SMA-CD34 morphologic grading, a CellProfiler image analysis pipeline was developed yielding a non-vessel associated normalized SMA area metric as a supervised correlate of the clinical SMA scoring system (R 2 = 0.68). Additional supervised and unsupervised bioinformatic approaches for clustering of relevant SMA-CD34 features including an algorithm that informs SMA spatial patterns with respect to niche elements such as arterioles (CD34+SMA+), sinusoids (CD34+) and adipocytes is in development.Lastly, Nanostring Fibrosis V2 panel was employed on a subset that met RNA concentration and quality metrics. Exploratory interpretation showed significant differentially expressed genes in pre vs. post JAKi specimens related to lipid metabolism such as ADIPOR1, SCD, ELOVL6 as well as the chemokine CXCL16. This may suggest a link between fatty acid metabolism and inflammatory differentiation along the SMA-vascular axis in the bone marrow modulated by JAKi treatment.SMA-CD34 IHC stratifies MF bone marrow biopsies differentially from standard WHO reticulin/trichome grading providing a practical formalin-fixed paraffin embedded (FFPE) tissue-based biomarker for assessing fibrosis related bone marrow niche elements from archived clinical samples. While our pilot numbers precluded statistical evaluation by JAKi-type, clinical response and NGS mutational profile at this time, further studies are underway to validate the SMA-CD34 signature on a larger MF cohort. [Display omitted] DisclosuresGupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; Incyte: Honoraria, Research Funding; Constellation Pharma: Consultancy, Honoraria; Pfizer: Consultancy.

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase β1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane “railroad track” structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

S3059 Clear Cell Sarcoma of the Jejunum: Dissecting an Obscure Malignancy

Introduction: Clear Cell Sarcoma (CCS) of the jejunum is an extremely rare and mortiferous presentation of a poorly understood malignancy. CCS is a cancer of the soft tissue and aponeuroses of tendons, affecting young individuals with predilection towards the ankle. It is often confused for malignant melanoma, as it is also derived from neural crest origin and shares a similar histological appearance. Currently, the only definitive treatment is surgical resection. Even so, CCS is known to recur. Due to the sheer modicum of cases, our medical knowledge is limited. Case Description/Methods: We present a case of a 49-year-old gentleman from Cameroon with a past medical history of hypertension and diabetes mellitus type II, who presented to the emergency department with complaints of abdominal pain, nausea, and vomiting for a one-month duration. CT scan of the abdomen and pelvis revealed left-sided small bowel obstruction. The patient underwent diagnostic laparoscopy, which converted to open surgery, with small bowel resection and umbilical hernia repair. A 2.6 cm circumferential obstructing jejunal mass was identified and resected. The margins of resection were negative for malignancy. There was no lymph vascular invasion, and 12 lymph nodes were negative for malignancy as well. Immunohistochemistry stains resulted positive for S100, SOX 10, and EMA. FISH was positive for EWSR1. Recent studies have revealed a chromosomal translocation, t(12;22) (q13;q12), also known as the EWSR1 and ATF1 gene, respectively, which has been widely accepted as pathognomonic for CCS. At 2 years of follow-up, our patient had a drastic improvement in his symptoms, with active surveillance and close follow-up, as CCS has a high recurrence rate. Discussion: This case report aims to describe the clinical presentation and evolution of this unique soft-tissue malignancy, which can be found in the GI tract in extraordinary cases. We hope this rare case of Clear Cell Sarcoma can enhance our knowledge about this obscure cancer that we still have so much to learn.Figure 1.: This 2.6 cm tumor is present in the submucosa, muscularis propria and subserosa and shows nests and cords of patterns by network of delicate vascular septa. Neoplastic cells are plump with epithelioid, clear cell and focal spindle cell appearance. Mitotic figures are about 5/10HPF. Immunostains show that tumor cells are positive for S100, SOX-10, and EMA but negative for HMB-45, Melan A, carcinoma markers (AE1/AE3, CAM5.2, CK7 and CK20), gastrointestinal stromal tumor markers (CKIT, CD34, and DOG1), lymphoid markers (CD3 and CD20), and desmin and ASMA. EWSR1 is positive by FISH studies. A) (Magnification x40, lower power, hematoxylin-eosin) and B) (Magnification x400, high power, hematoxylin-eosin) show epithelioid and clear cell appearance. C) (Magnification x400, antibody for S100) and D) (Magnification x400, antibody for SOX-10) reveal positivity.

Identification of Predictive Markers for the Generation of Well-Differentiated Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Human-induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential to advance studies to kidney development and disease. However, reproducible generation of kidney organoids is a challenge. A large variability in the percentage of nephron structures and the expression of kidney-specific genes was observed among organoids, showing no association with iPSC lines. To associate the quality of kidney organoid differentiation with predictive markers, a ranking system was developed based on the ratio of nephron structure determined by histological examination. Well-differentiated organoids were defined as organoids with >30% nephron structure and vice versa. Subsequently, correlations were made with expression profiles of iPSC markers, early kidney development markers, and fibrosis markers. Higher expression of sex-determining region Y-box 2 (SOX2) during differentiation was associated with poorly differentiated kidney organoid. Furthermore, early secretion of basic fibroblast growth factor (FGF2) predicted poorly differentiated kidney organoid. Of interest, whereas cadherin-1 (CDH1) expression in kidney organoids indicates distal tubules formation, onefold higher CDH1 expression in iPSC predicted poor differentiation. High expression of the stromal progenitor marker Forkhead Box D1 (FOXD1) and significantly increased TGFβ levels were found in well-differentiated kidney organoids. These early expression profiles could predict the outcome of kidney organoid formation. This study helps to improve the robustness of kidney organoid protocols.

Stromal marker fibroblast activation protein drives outcome in T1 non-muscle invasive bladder cancer.

Fibroblast activation protein-α (FAP) is a transmembrane peptidase and a surrogate marker for cancer-associated fibroblasts (CAFs). FAP has been linked to worse prognosis and therapy resistance in several cancers. We hypothesised that FAP might have a prognostic 3biomarker potential to stratify patients with high-grade (HG) T1 non-muscle-invasive bladder cancer (NMIBC). We selected 30 patients with HG T1 NMIBC that progressed to ≥T2 disease which were pair-matched based on CUETO progression score variables with 90 patients that did not progress. After revision a final cohort of 86 patients was retained. Slides were stained for FAP, the luminal marker GATA3 and the basal marker CK5. All HG T1 tumour regions of interest (ROIs) within each patient were annotated, analysed and scored using image analysis software. FAP expression in HG T1 ROIs was significantly higher in progressors vs. non-progressors and was prognostic for recurrence-free survival, progression-free survival, cancer-specific survival, and overall survival. FAP expression in HG T1 ROIs remained strongly prognostic for these outcomes in a bivariable model corrected for adequate BCG per FDA definition. Expression of GATA3 and CK5 did not differ between progressors vs. non-progressors, and were not prognostic for these outcomes. FAP might serve as an easily applicable prognostic biomarker to risk-stratify patients with HG T1 NMIBC if these results are prospectively validated in a larger series.