The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC

Durable complete molecular remission of chronic myeloid leukemia following dasatinib cessation, despite adverse disease features

Patients with chronic myeloid leukemia, treated with imatinib, who have a durable complete molecular response, might remain in complete molecular response after stopping treatment. Previous reports of patients stopping treatment in complete molecular response have included only patients with a good response to imatinib. We describe 3 patients with stable complete molecular response on dasatinib treatment following imatinib failure. Two of the 3 patients remain in complete molecular response more than 12 months after stopping dasatinib. In these 2 patients we used highly sensitive patient-specific BCR-ABL1 DNA PCR to show that the leukemic clone remains detectable, as we have previously shown in imatinib-treated patients. Dasatinib-associated immunological phenomena, such as the emergence of clonal T-cell populations, were observed both in one patient who relapsed and in one patient in remission. Our results suggest that the characteristics of complete molecular response on dasatinib treatment may be similar to that achieved with imatinib, at least in patients with adverse disease features

Chemical inhibition of ABCC6 increases the efficacy of dasatinib but not imatinib in patient MNCs.

<p>p-CRKL determined IC50 was determined via incubating patient MNCs with increasing concentrations of (A) dasatinib and (B) imatinib in the absence and presence of three ABCC6 inhibitors: pantoprazole, indomethacin, probenecid. CRKL western blotting was performed to determine the concentration of TKI required for 50% Bcr-Abl kinase inhibition (IC50). Representative western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s004" target="_blank">S4</a> Figs. The box plots depict the median, the upper 25th and the lower 75th percentiles while the whiskers encompass the 10th and 90th percentiles. Statistical analyses were performed using Student’s <i>t</i>-test with statistically <i>p</i>-values denoted by asterisks (** <i>p</i><0.01; *** <i>p</i><0.001). IM = imatinib. DAS = dasatinib.</p

Chemical inhibition of ABCC6 increases the efficacy of dasatinib but not imatinib in <i>BCR-ABL1</i>+ cell lines.

<p>(A,E) K562 (B,F) K562-Dox (C,G) K562-ABCG2 and (D,H) KU812 cells were incubated with the indicated concentrations of (A-D) dasatinib and (E-H) imatinib in the absence and presence of three ABCC6 inhibitors (200 μM pantoprazole, 100 μM indomethacin, 1 mM probenecid). CRKL western blotting was performed to determine the concentration of TKI required for 50% Bcr-Abl kinase inhibition (IC50). Error bars represent SEM. Representative western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s004" target="_blank">S4</a> Figs. IM = imatinib; DAS = dasatinib; PP = pantoprazole; Indo = Indomethacin; Pro = probenecid.</p

The effect of increasing concentrations of pantoprazole on IC50^NIL in <i>BCR-ABL1</i>+ cell lines.

<p>The effect of increasing concentrations of pantoprazole on IC50^NIL in <i>BCR-ABL1</i>+ cell lines.</p

Chemical inhibition of ABCC6 increases the efficacy of nilotinib in <i>BCR-ABL1</i>+ cell lines.

<p>(A) Expression levels of ABCC6 protein were assessed in K562, K562-Dox, K562-ABCG2 and KU812 <i>BCR-ABL1</i>+ cell lines. The western blot analyses are representative and the corresponding densitometry analysis represents the mean of three experiments. HepG2 cells were used as a positive control and protein levels were normalised to β-ACTIN. HepG2 cells express two splice variants of ABCC6; <i>BCR-ABL1</i>+ cell lines express the N-glycosylated isoform only. (B) K562 (C) K562-Dox (D) K562-ABCG2 and (E) KU812 cells were incubated with increasing concentrations of nilotinib in the absence and presence of three ABCC6 inhibitors (200 μM pantoprazole, 100 μM indomethacin, 1 mM probenecid). CRKL western blotting was performed to determine the concentration of TKI required for 50% Bcr-Abl kinase inhibition (IC50^NIL). Two K562 control bars are shown as experiments in the two groups were performed months apart. Error bars represent SEM. Representative western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s002" target="_blank">S2 Fig</a>. NIL = nilotinib; PP = pantoprazole; Indo = Indomethacin; Pro = probenecid.</p

<i>ABCB1</i> and <i>ABCC6</i> mRNA levels increase in concert during development of nilotinib resistance in <i>BCR-ABL1</i>+ cell lines.

<p>(A) Expression levels of <i>ABCC6</i> mRNA were assessed by Taqman^® transporter array in K562 and KU812 cells exposed transiently (overnight, O/N) and long term to nilotinib Expression levels of <i>ABCC6</i> and <i>ABCB1</i> mRNA were assessed in (B) K562 and (C) K562-Dox cells gradually made resistant to nilotinib by exposure to increasing concentrations over time. (A) <i>ABCC6</i> levels were normalized to selected control genes (as determined by Thermo Fisher Scientific DataAssist Software v1.0) and fold change in cells cultured in the presence of nilotinib calculated relative to cells cultured in the absence of nilotinib. (B-C) <i>ABCC6</i> and <i>ABCB1</i> levels were normalized to the housekeeping gene <i>BCR</i> and fold change in resistance intermediates calculated relative to control cells (control cell fold change was set at 1). The mRNA expression represents the mean of six independent experiments performed in triplicate. Statistical analyses were performed using Student’s <i>t</i>-test. Statistically significant <i>p</i>-values are denoted by asterisks (ABCC6) and carets (ABCB1) * p<0.05; ** p<0.01; *** p<0.001). Error bars represent SEM. NIL = nilotinib; RES = resistant.</p

Chemical inhibition of ABCC6 but not ABCB1 increases the efficacy of nilotinib in patient MNCs.

<p>p-CRKL determined IC50^NIL was determined via incubating patient MNCs with increasing concentrations of nilotinib in the absence and presence of (A) three ABCB1 inhibitors: PSC-833, verapamil and pantoprazole and (B) three ABCC6 inhibitors: pantoprazole, indomethacin, probenecid. CRKL western blotting was performed to determine the concentration of nilotinib required for 50% Bcr-Abl kinase inhibition (IC50^NIL). Representative western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192180#pone.0192180.s001" target="_blank">S1 Fig</a>. The box plots depict the median, the upper 25th and the lower 75th percentiles while the whiskers encompass the 10th and 90th percentiles. Statistical analyses were performed using Student’s <i>t</i>-test with statistically significant <i>p</i>-values denoted by asterisks (*** p<0.001). NIL = nilotinib.</p