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Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

No MeSH data available.


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Analysis of impaired pancreatic cancer growth with shMSI and MSI1-ASOs(a) Schematic for inhibiting MSI in primary patient-derived xenografts. (b-c) Frequency of GFP+ patient tumor cells before and after transplantation. See also Figure 4a-b for Patient#1, #2. ASO delivery in vivo(d, e) MSI1 expression following free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n=3 per condition). See also Figure 4c for impact of MSI1-ASO1. Target knockdown efficacy of lead optimized ASO in KPf/fC stem cells (f) Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH- cells following systemic delivery of lead-optimized control ASO or Malat1-ASO in autocthonous KPf/fC model (n=3 independent experiments) See also Figure 4h for target knockdown in unfractionated Epcam+ cells. Analysis of potential toxicity of MSI-ASO (g) Cage weight of mice receiving daily treatment of MSI1 ASO-1 (50mg/kg) or vehicle by IP injection; 4 mice per cage; cage weight was measured every 3 days. (h) Average body weight of mice following 3 weeks of daily treatment with MSI1 ASO-1 (50mg/kg) or vehicle by IP injection (n=4 mice/cohort). In vivo delivery of MSI1 ASOs (50mg/kg) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3x ULN (upper limit of normal). (i-j) Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumors isolated from KPf/fC mice treated by daily IP injection with (i) control ASO (50mg/kg) or (j) Malat1-ASO (50mg/kg) for 14 days. Source Data for all panels are available online.
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Figure 12: Analysis of impaired pancreatic cancer growth with shMSI and MSI1-ASOs(a) Schematic for inhibiting MSI in primary patient-derived xenografts. (b-c) Frequency of GFP+ patient tumor cells before and after transplantation. See also Figure 4a-b for Patient#1, #2. ASO delivery in vivo(d, e) MSI1 expression following free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n=3 per condition). See also Figure 4c for impact of MSI1-ASO1. Target knockdown efficacy of lead optimized ASO in KPf/fC stem cells (f) Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH- cells following systemic delivery of lead-optimized control ASO or Malat1-ASO in autocthonous KPf/fC model (n=3 independent experiments) See also Figure 4h for target knockdown in unfractionated Epcam+ cells. Analysis of potential toxicity of MSI-ASO (g) Cage weight of mice receiving daily treatment of MSI1 ASO-1 (50mg/kg) or vehicle by IP injection; 4 mice per cage; cage weight was measured every 3 days. (h) Average body weight of mice following 3 weeks of daily treatment with MSI1 ASO-1 (50mg/kg) or vehicle by IP injection (n=4 mice/cohort). In vivo delivery of MSI1 ASOs (50mg/kg) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3x ULN (upper limit of normal). (i-j) Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumors isolated from KPf/fC mice treated by daily IP injection with (i) control ASO (50mg/kg) or (j) Malat1-ASO (50mg/kg) for 14 days. Source Data for all panels are available online.

Mentions: To complement the mouse models, we tested the impact of MSI inhibition on primary patient samples, which harbor more complex mutations, and are uniformly drug resistant. Primary pancreatic cancer cells were infected with MSI shRNAs and xenografted (Extended Data Fig. 8a). While shMSI cells were equivalently present at time of transplant, their ability to contribute to the tumor mass in vivo was reduced by 4.9-6.5 fold (Fig. 4a-b, Extended Data Fig. 8b-c), demonstrating that inhibition of either MSI1 or MSI2 results in marked suppression of primary human pancreatic cancer growth. Interestingly, MSI2 expression was more homogeneous in patients than in mouse models (Extended Data Fig. 1a-b, 2d-e). This could be a consequence of selection due to treatment and end-stage disease in patients, or because MSI2 patterns differ between mouse models and human disease. However, regardless of the level of heterogeneity, our loss-of-function studies indicate that the mouse and human disease are both highly dependent on Msi signaling.


Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma
Analysis of impaired pancreatic cancer growth with shMSI and MSI1-ASOs(a) Schematic for inhibiting MSI in primary patient-derived xenografts. (b-c) Frequency of GFP+ patient tumor cells before and after transplantation. See also Figure 4a-b for Patient#1, #2. ASO delivery in vivo(d, e) MSI1 expression following free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n=3 per condition). See also Figure 4c for impact of MSI1-ASO1. Target knockdown efficacy of lead optimized ASO in KPf/fC stem cells (f) Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH- cells following systemic delivery of lead-optimized control ASO or Malat1-ASO in autocthonous KPf/fC model (n=3 independent experiments) See also Figure 4h for target knockdown in unfractionated Epcam+ cells. Analysis of potential toxicity of MSI-ASO (g) Cage weight of mice receiving daily treatment of MSI1 ASO-1 (50mg/kg) or vehicle by IP injection; 4 mice per cage; cage weight was measured every 3 days. (h) Average body weight of mice following 3 weeks of daily treatment with MSI1 ASO-1 (50mg/kg) or vehicle by IP injection (n=4 mice/cohort). In vivo delivery of MSI1 ASOs (50mg/kg) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3x ULN (upper limit of normal). (i-j) Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumors isolated from KPf/fC mice treated by daily IP injection with (i) control ASO (50mg/kg) or (j) Malat1-ASO (50mg/kg) for 14 days. Source Data for all panels are available online.
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Related In: Results  -  Collection

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Figure 12: Analysis of impaired pancreatic cancer growth with shMSI and MSI1-ASOs(a) Schematic for inhibiting MSI in primary patient-derived xenografts. (b-c) Frequency of GFP+ patient tumor cells before and after transplantation. See also Figure 4a-b for Patient#1, #2. ASO delivery in vivo(d, e) MSI1 expression following free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n=3 per condition). See also Figure 4c for impact of MSI1-ASO1. Target knockdown efficacy of lead optimized ASO in KPf/fC stem cells (f) Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH- cells following systemic delivery of lead-optimized control ASO or Malat1-ASO in autocthonous KPf/fC model (n=3 independent experiments) See also Figure 4h for target knockdown in unfractionated Epcam+ cells. Analysis of potential toxicity of MSI-ASO (g) Cage weight of mice receiving daily treatment of MSI1 ASO-1 (50mg/kg) or vehicle by IP injection; 4 mice per cage; cage weight was measured every 3 days. (h) Average body weight of mice following 3 weeks of daily treatment with MSI1 ASO-1 (50mg/kg) or vehicle by IP injection (n=4 mice/cohort). In vivo delivery of MSI1 ASOs (50mg/kg) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3x ULN (upper limit of normal). (i-j) Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumors isolated from KPf/fC mice treated by daily IP injection with (i) control ASO (50mg/kg) or (j) Malat1-ASO (50mg/kg) for 14 days. Source Data for all panels are available online.
Mentions: To complement the mouse models, we tested the impact of MSI inhibition on primary patient samples, which harbor more complex mutations, and are uniformly drug resistant. Primary pancreatic cancer cells were infected with MSI shRNAs and xenografted (Extended Data Fig. 8a). While shMSI cells were equivalently present at time of transplant, their ability to contribute to the tumor mass in vivo was reduced by 4.9-6.5 fold (Fig. 4a-b, Extended Data Fig. 8b-c), demonstrating that inhibition of either MSI1 or MSI2 results in marked suppression of primary human pancreatic cancer growth. Interestingly, MSI2 expression was more homogeneous in patients than in mouse models (Extended Data Fig. 1a-b, 2d-e). This could be a consequence of selection due to treatment and end-stage disease in patients, or because MSI2 patterns differ between mouse models and human disease. However, regardless of the level of heterogeneity, our loss-of-function studies indicate that the mouse and human disease are both highly dependent on Msi signaling.

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus