Figure S5 from Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Supplemental Figure S5. Metastatic seeding of BMP7 treated cells is not enhanced by neutrophil depletion in tumor baring mice. Representative images of lung associated GFP+ BMP7 treated cells in control (BMP7) and neutrophil depleted (BMP7 depletion) mice, 48 hrs following tail-vein injection.</p

Figure S2 from Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Supplemental Figure S2. Circulating tumor cells express higher levels of TRPM2 compared with primary tumor cells in pancreatic cancer patients. A. Transcriptome analysis of mesenchymal genes, epithelial genes and TRPM2 on primary 4T1 tumor cells and circulating 4T1 tumor cells. The analysis was performed on dataset (GSE37244) published by LeBlue et al. (28). B. Microarray study for gene expression (GSE18670) changes in 6 samples from pancreatic cancer primary tumor (Primary) and circulating tumor cells (CTC) was described previously by Sergeant at al. (29). Here the same dataset was reanalyzed for the log2 expression ratio of TRMP2 in these samples. Data show TRPM2 expression in primary tumor cells (blue) and CTC (Red). *p<0.05 (2-tailed paired t-test).</p

Figure S3 from Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Supplemental Figure S3. Inverse correlation between E-Cadherin expression and TRPM2 expression in primary and metastatic 4T1 tumors. Immunofluorescent staining of TRPM2 (red) and E-Cadherin (green) in 4T1 primary tumor (A) in a lung metastatic 4T1 tumor (B).</p

Figure S4 from Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Supplemental Figure S4. EMT increases TRPM2 expression and renders cells susceptible to neutrophil cytotoxicity. A. Representative images showing control and Snail overexpressing 4T1 cells. B. Snail expression in control and Snail overexpressing 4T1 cells. C. qPCR analysis of TRPM2, Vimentin (Vim.) and E-Cadherin (E-Cad.) expression in control and Snail overexpressing 4T1 cells. D. Extent of neutrophil cytotoxicity in co-culture with 4T1 control cells (Cont.) or 4T1 cells induced to undergo EMT by Snail overexpression (Snail). E. Representative images showing HMLE cells before (HMLE) and after prolonged treatment with 10 ng/ml TGFβ to induce EMT. F. qPCR analysis of TRPM2, Vimentin (Vim.) and E-Cadherin (E-Cad.) in control and TGFβâ^'treated HMLE cells. G. Extent of neutrophil cytotoxicity in co-culture with control (Cont.) or TGFβ-pretreated HMLE cells that have undergone EMT. H. Representative images showing control and Twist overexpressing HMLE cells. I. Twist expression in control and Twist overexpressing HMLE cells. J. qPCR analysis of TRPM2, Vimentin (Vim.) and E-Cadherin (E-Cad.) expression in control and Twist overexpressing HMLE cells. K. Extent of neutrophil cytotoxicity in co-culture with HMLE control cells (Cont.) or HMLE cells induced to undergo EMT by Twist overexpression (Twist). *p<0.05, **p<0.01.</p

Figure S1 from Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Supplemental Figure S1. Neutrophils in the premetastatic lung outnumber and interact with disseminated tumor cells. Ly6G immunohistochemistry of metastases-laden lungs from 28 day 4T1 tumor-bearing mouse. Insets are high power magnification.</p

Image_2_2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.tif

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as “natural” drug delivery systems for targeting specific cell types.</p

Image_3_2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.tif

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as “natural” drug delivery systems for targeting specific cell types.</p

Image_4_2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.tif

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as “natural” drug delivery systems for targeting specific cell types.</p

Image_6_2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.tif

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as “natural” drug delivery systems for targeting specific cell types.</p

Image_7_2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.tif

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as “natural” drug delivery systems for targeting specific cell types.</p