IntroductionColorectal cancer (CRC) is a heterogeneous malignancy and a major cause of cancer-related mortality worldwide. Cancer-associated fibroblasts (CAFs) accumulate in tumors and correlate with poor patient survival, suggesting a central role in immune regulation. Patient-derived organoids (PDO) maintain the intra-tumoral cellular heterogeneity of the original tissue, thus, they represent one of the best methods to study human cancers. The tumor microenvironment (TME) contains diverse immune cell populations, including innate lymphoid cells (ILCs), yet how stromal components influence cytokine-driven tumor–immune interactions remains unclear.MethodsPDOs from CRC patients were used to screen TME-derived cytokines affecting tumor growth. Organoid-forming efficiency and signaling pathway activation were analyzed following cytokine stimulation in the presence or absence of CAFs. Tumor-infiltrating ILC subsets were characterized in patient samples, and co-culture systems were employed to assess cytokine production and stromal–immune interactions.ResultsIL-22 was identified as a cytokine that increased PDO-forming efficiency without activating fibroblasts. Although IL-22 typically signals through the JAK–STAT pathway, it unexpectedly activates MAPK signaling in PDO cells. Interestingly, interferon-γ (IFNγ) showed only partial cytotoxic effects on CRC cells. Tumor tissues contained both IFNγ-producing ILC1 and IL-22/IL-17A–producing ILC3 populations. Co-culture with PDOs selectively induced IL-22, but not IL-17A, production in ILC3 cells. Importantly, IL-22 enhanced organoid formation only in the absence of CAFs, whereas IFNγ activity was largely unaffected by stromal context.DiscussionThese findings demonstrate that CAFs modulate local immunity by selectively masking ILC3-derived IL-22 signaling while preserving ILC1-mediated IFNγ responses. This study emphasizes the importance of stromal context in interpreting cytokine function in CRC and reveals a previously unrecognized mechanism of stromal–immune crosstalk within the TME.

Colorectal cancer (CRC) is the second-third most frequent cancer type in the Western countries, with significant inter- and intra-patient cellular heterogeneity. The transcriptomics-based CRC classification identified a cluster of patients with epithelial-mesenchymal transition (EMT), the accumulation of fibroblasts, and dismal prognosis. Although the serotonin receptor HTR2B is thought to be one of the epithelial markers of this aggressive CMS4 subgroup, its precise role in CRC tumorigenesis is still largely unknown. By using patient-derived organoids (PDO), we prove the heterogeneous expression of this receptor that is under the control of the mTOR pathway. Unfavorable conditions, applying the clinically used 5-fluorouracil, co-culturing with fibroblasts, and collagen-I accumulation in the extracellular matrix (ECM) increased the number of HTR2B + cells. Interestingly, whereas stimulating HTR2B had no effect on PDOs under normal conditions, it reduced cellular survival under unfavorable conditions when carbohydrates were lacking. In contrast, serotonin and an HTR2B agonist induced invasion of HTR2B + tumor cells only in collagen-I, representing a permissive ECM for cellular migration. CRC cells positive for HTR2B also expressed the NOTCH3 receptor and NOTCH target genes at a higher intensity than cells with low HTR2B levels. Similar to HTR2B + organoids, NOTCH3 + PDOs had increased EMT markers and HTR2B levels. Furthermore, blocking NOTCH activity inhibited the serotonin-induced invasion. Collectively, our results show that the serotonin receptor HTR2B marks a CRC cell population with increased proliferative capacity, invasive potential, and partial EMT, and they highlight the critical role of ECM in shaping the effect of serotonin in CRC.

Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles secreted by most cells into the biofluids. They have an important role in intercellular communication by carrying bioactive components (e.g., lipids, proteins, nucleic acids, and other metabolites) and reflecting the biochemical and metabolic processes of their parent cell. Due to their widespread molecular cargo, they have potential diagnostic and therapeutic roles, so they can be considered as new generation biomarkers. With the drastic increase in the number of publications and the comparability of the obtained results, the development of standardized protocols has become necessary. Here we aim to demonstrate the applicability of two, non-conventional techniques, such as ATR-infrared spectroscopy (ATR-FTIR) and electromagnetic piezoelectric acoustic sensor (EMPAS) in the characterization of compositional changes of blood- and pancreatic tumor-derived vesicles. In addition, we provide guidelines in sample collection, isolation, and separation of EVs. IR spectroscopy, as a fast and label-free technique, gives biochemical insight into the sample composition, while EMPAS serves information on vesicle quantity and in membrane integrity changes during storage.

PDF file - 1.8MB, Effect of homozygous or haploid Bmx deletion on tumor growth in female mice in the chemical skin carcinogenesis model

PDF file - 196K, Normal postnatal development of retinal vasculature in Bmx deficient mice

Supplementary Figure Legend 2 from Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

Colorectal cancer (CRC) has a high incidence and is one of the leading causes of cancer-related death. The accumulation of cancer-associated fibroblasts (CAF) induces an aggressive, stem-like phenotype in tumor cells, and it indicates a poor prognosis. However, cellular heterogeneity among CAFs and the targeting of both stromal and CRC cells are not yet well resolved. Here, we identified CD142high fibroblasts with a higher stimulating effect on CRC cell proliferation via secreting more hepatocyte growth factor (HGF) compared to CD142low CAFs. We also found that combinations of inhibitors that had either a promising effect in other cancer types or are more active in CRC compared to normal colonic epithelium acted synergistically in CRC cells. Importantly, heat shock protein 90 (HSP90) inhibitor selected against CD142high fibroblasts, and both CRC cells and CAFs were sensitive to a BCL-xL inhibitor. However, targeting mitogen-activated protein kinase kinase (MEK) was ineffective in fibroblasts, and an epigenetic inhibitor selected for a tumor cell population with markers of aggressive behavior. Thus, we suggest BCL-xL and HSP90 inhibitors to eliminate cancer cells and decrease the tumor-promoting CD142high CAF population. This may be the basis of a strategy to target both CRC cells and stromal fibroblasts, resulting in the inhibition of tumor relapse.

PDF file - 93K, No effect on tumor growth in the Bmx-deleted male mice in the chemically induced skin carcinogenesis model or RIP1-Tag2 pancreatic insulinomas

Supplementary Figure Legend 3 from Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

Supplementary Table from Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

Supplementary Figure 1 from Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

Extracellular vesicles (EV) carry their cargo in a membrane protected form, however, their value in early diagnostics is not well known. Although pancreatic cysts are heterogeneous, they can be clustered into the larger groups of pseudocysts (PC), and serous and mucinous pancreatic cystic neoplasms (S-PCN and M-PCN, respectively). In contrast to PCs and S-PCNs, M-PCNs may progress to malignant pancreatic cancers. Since current diagnostic tools do not meet the criteria of high sensitivity and specificity, novel methods are urgently needed to differentiate M-PCNs from other cysts. We show that cyst fluid is a rich source of EVs that are positive and negative for the EV markers CD63 and CD81, respectively. Whereas we found no difference in the EV number when comparing M-PCN with other pancreatic cysts, our EV-based biomarker identification showed that EVs from M-PCNs had a higher level of miR-200b. We also prove that not only EV-derived, but also total cyst fluid miR-200b discriminates patients with M-PCN from other pancreatic cysts with a higher sensitivity and specificity compared to other diagnostic methods, providing the possibility for clinical applications. Our results show that measuring miR-200b in cyst fluid-derived EVs or from cyst fluid may be clinically important in categorizing patients.

Extracellular vesicles (EV) are released by virtually all cells and they transport biologically important molecules from the release site to target cells. Colorectal cancer (CRC) is a leading cause of cancer-related death cases, thus, it represents a major health issue. Although the EV cargo may reflect the molecular composition of the releasing cells and thus, EVs may hold a great promise for tumor diagnostics, the impact of intratumoral heterogeneity on the intensity of EV release is still largely unknown. By using CRC patient-derived organoids that maintain the cellular and molecular heterogeneity of the original epithelial tumor tissue, we proved that CD44high cells produce more organoids with a higher proliferation intensity, as compared to CD44low cells. Interestingly, we detected an increased EV release by CD44high CRC cells. In addition, we found that the miRNA cargos of CD44high and CD44low cell derived EVs largely overlapped and only four miRNAs were specific for one of the above subpopulations. We observed that EVs released by CD44high cells induced the proliferation and activation of colon fibroblasts more strongly than CD44low cells. However, this effect was due to the higher EV number rather than to the miRNA cargo of EVs. Collectively, we identified CRC subpopulations with different EV releasing capabilities and we proved that CRC cell-released EVs have a miRNA-independent effect on fibroblast proliferation and activation.

The majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which leads to an unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered as potential therapeutic tools. Although CRC is a genetically heterogenous disease, the significance of the inter- and intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induces the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1 high CRC cells contained more proliferating cells. Whereas we found no difference in the uptake of liposomes between IFITM1 high and IFITM1 low/− cells, IFITM1 high cells had a markedly reduced uptake of fibroblast EVs as compared to IFITM1 low/− cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 level. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1 low CRC cells. Collectively, we identified CRC cell subpopulations with functional difference in their EV, but not liposome uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.

Extracellular vesicles (EV) are considered as a potential tool for early disease diagnosis; however, factors modifying EV release remain partially unknown. By using patient-derived organoids that capture the cellular heterogeneity of epithelial tissues, here we studied the connection between the Wnt-producing microniche and EV secretion in multiple tissues. Although nearly all cells in pancreatic ductal (PD) and pancreatic ductal adenocarcinoma (PDAC) samples expressed porcupine (PORCN), an enzyme critical for Wnt secretion, only a subpopulation of lung bronchiolar (NL) and lung adenocarcinoma (LUAD) organoid cells produced active Wnt. The microniche for proliferating cells was shaped not only by PORCN + cells in NL and LUAD organoids but also by fibroblast-derived EVs. This effect could be blocked by using Wnt secretion inhibitors. Whereas inhibiting Wnt secretion in PD NL or LUAD organoids critically changed both cell proliferation and EV release, these were uncoupled from each other in PDAC. Sorting for CD133 identified a cell population in the LUAD microniche that produced organoids with a high percentage of PORCN + and proliferating cells and an elevated EV secretion, which may explain that CD133 marks LUAD cells with malignant behavior. Collectively, we show here that high cell proliferation rate, induced by Wnt pathway activation, is coupled to a higher EV release, a critical finding that may be considered when developing EV-based diagnostic tools.

The majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which lead to the unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered potential therapeutic tools. Although CRC is a genetically heterogeneous disease, the significance of the intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induced the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1high CRC cells contained more proliferating cells and they had a markedly reduced uptake of fibroblast EVs as compared to IFITM1low/− cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 levels. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1low CRC cells. Furthermore, inactivating IFITM1 resulted in an enhanced EV uptake, highlighting the importance of this molecule in establishing the cellular difference for EV effects. Collectively, we identified CRC cells with functional difference in their EV uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.

Extracellular vesicles (EV), structures surrounded by a biological membrane, transport biologically active molecules, and represent a recently identified way of intercellular communication. Colorectal cancer (CRC), one of the most common cancer types in the Western countries, is composed of both tumor and stromal cells and the amount of stromal fibroblasts negatively correlates with patient survival. Here we show that normal colon fibroblasts (NCF) release EVs with a characteristic miRNA cargo profile when stimulated with TGFβ, one of the most important activating factors of fibroblasts, without a significant increase in the amount of secreted EVs. Importantly, fibroblast-derived EVs induce cell proliferation in epidermal growth factor (EGF)-dependent patient-derived organoids, one of the best current systems to model the intra-tumoral heterogeneity of human cancers. In contrast, fibroblast-derived EVs have no effect in 3D models where EGF is dispensible. This EV-induced cell proliferation did not depend on whether NCFs or cancer-associated fibroblasts were studied or on the pre-activation by TGFβ, suggesting that TGFβ-induced sorting of specific miRNAs into EVs does not play a major role in enhancing CRC proliferation. Mechanistically, we provide evidence that amphiregulin, transported by EVs, is a major factor in inducing CRC cell proliferation. We found that neutralization of EV-bound amphiregulin blocked the effects of the fibroblast-derived EVs. Collectively, our data suggest a novel mechanism for fibroblast-induced CRC cell proliferation, coupled to EV-associated amphiregulin.

Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.

Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblast-derived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche.