The human HPV positive HNSCC cell line UPCI: SCC154 was purchased from American Type Culture Collection (#CRL-3241, ATCC) and UD-SCC-2 (RRID: CVCL_E325) was obtained from the Dr. Silvio Gutkind’s Laboratory at the Moores Cancer Center, University of California San Diego. UPCI: SCC154 is a HPV positive HNSCC cell line derived from a squamous cell carcinoma of the tongue of a 54 year old white male. UD-SCC-2 is also a HPV positive HNSCC cell line derived from squamous cell carcinoma of the hypopharynx of a 58 year old male. These cell lines are well characterized HPV positive HNSCC cell lines (34–37). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus penicillin (50 U/ml) and streptomycin (50 μg/ml). The mouse tonsil epithelial cells expressing HPV-16 E6 and E7 and H-Ras (mEER) (38–40) were cultured in E-Media containing 67% DMEM (Fisher Scientific), 10% FBS (Invitrogen), 22% Ham’s F-12 Nutrient Mix (Fisher Scientific), 1% penicillin/streptomycin (Invitrogen), 25µg/ml hydrocortisone (Sigma), 5µg/ml insulin (Sigma), 5µg/ml transferrin (Sigma), 1.36ng/ml tri-iodo-thyronine (Sigma), 8.4ng/ml cholera toxin (Sigma) and 5ng/ml epidermal growth factor (Invitrogen). The cells were maintained at 37°C under an atmosphere of 5% CO₂.

The proliferation of HPV-positive HNSCC cells UD-SCC-2 and UPCI: SCC154 upon treatment with Cannabidiol (CBD) (Cayman Chemicals) was determined by using AquaBluer Solution (MultiTarget Pharmaceuticals). Briefly, 1 x 10⁵ cells/well were seeded overnight into 96-well plates and the next day treated with 10μM of CBD and Vehicle (1:9 DMSO: PBS) as the control for 24 and 48 hours. The cells were stained with 1X AquaBluer Solution for 3 hours at 37°C and fluorescence intensity was measured at 540nm for excitation and 580nm for emission by using a microplate reader (TECAN Spark).

Furthermore, the incorporation of 5-bromo-2’-deoxyuridine (BrdU) into cellular DNA during cell proliferation was measured using an anti-BrdU antibody in HPV-positive HNSCC cells UD-SCC-2 and UPCI: SCC154. Cells were seeded at a density of 1 × 10⁵ cells/well into 96-well microtiter plates overnight followed by treatment with 10μM of CBD for 24 and 48 hours. After incubation with drugs, the cells were incubated with 1X BrdU for 24 h. The BrdU assay kit was purchased from Cell Signaling Technology (Danvers, MA, USA). BrdU assay was carried out as described previously by Sen et al. (41). The intensity was measured at 450 nm using a microplate reader (TECAN Spark). The percentage of cell proliferation was calculated as relative to that of the control values using GraphPad Prism 10.4.0 software.

The HPV-positive HNSCC cell lines UPCI: SCC154 and UD-SCC-2 were used to determine apoptosis using APC Annexin V Apoptosis Detection Kit with PI (BioLegend, San Diego, CA, USA) after treatment with 10μM of CBD. Briefly, 1×10⁵ cells/well were plated into 12-well plates followed by treatment with 10μM of CBD for 24h and 48h. Cells were stained according to the manufacturer’s instructions and were analyzed using the NovoCyte Flow Cytometer Systems (Agilent Technologies). Data analysis was conducted using FlowJo (FlowJo, LLC).

A scratch or wound healing assay was performed to study cellular migration after treatment with 10μM of CBD in HPV-positive cell lines UD-SCC-2 and UPCI: SCC154 as reported earlier (41). The images of the wound were captured at 0h, 12h and 24 h using an inverted microscope (ECLIPSE Ts2R, Nikon) at 4X magnification. The images were analyzed using Image J software ver. 1.54 (NIH, Bethesda, MD, USA). The percentage of cell migration was calculated relative to the control using GraphPad Prism 10.4.0 software.

Western Blot was carried out for HPV-positive HNSCC cells UD-SCC-2 and UPCI: SCC154, post-treatment with CBD 1μM, 5μM, and 10μM for 15 and 30 minutes as reported earlier (24). The primary antibodies p38 (#8690), phospho- p38 (pp38) (#4511), Erk1/2 (#4370), phospho-extracellular signal-regulated kinase (pERK1/2), (#4695), SAPK/JNK (#9252), phospho-c-Jun N-terminal kinase (pJNK) (#4688), MAPKAPK-2 (#3042) and phospho-mitogen-activated-protein-kinase-activated-protein-kinase-2 (pMK-2) (#3007) and Vinculin (#4650) were obtained from Cell Signaling Technology (Danvers, MA, USA) and used at a dilution of 1:1000. Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling Technology (Danvers, MA, USA)) was used as the secondary antibody at a dilution of 1:2000. The blots were developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma, Burlington, MA, USA) and images were captured by iBright FL1500 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Vinculin was used as loading control. The western blot images were analyzed using the Image J software ver. 1.54 (NIH, Bethesda, MD, USA). The relative protein expression was measured with respect to control after normalizing with loading control (Vinculin). The ratios for pp38:p38, pERK1/2:ERK1/2, pSAPK: SAPK and pMK2:MK2 were measured after normalizing the band intensity of the respective protein markers with Vinculin. The original blots can be found in Supplementary Figures S2A-D .

Six weeks old male mice (wild type C57BL/6J mice, athymic nude mice and B6.129S7-Rag1^tm1Mom/J or Rag1 -/- also commonly known as Rag1 Knockout (Rag 1KO)) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). HPV-positive mEER cells, which are immortalized with a retrovirus containing HPV16 E6, HPV16 E7, and H-Ras genes (39, 40, 42), were used at a concentration of 1x10⁶ cells/mouse in 100μl of 1X PBS to develop subcutaneous tumors in the in the flank region of wild-type C57BL/6J mice, athymic nude mice which are immunodeficient, and Rag1 KO mice which do not produce mature T cells or B cells. Palpable tumors in the flank were observed 5 days after injection of cells and the mice were divided randomly into two groups. Next day (Day 6), the wild-type C57BL/6J mice, the athymic nude mice and Rag1 KO mice were treated with 10μM CBD and control vehicle (1:9 DMSO: PBS) intraperitoneally (I.P) every day and continued till Day 21. In addition, for CD4+T cells and CD8+T cells depletion in wild-type C57BL/6J mice, 1x10⁶ mEER cells/mouse was subcutaneously injected in the flank region of each of the mice. Mice were injected with 200 µg of anti-CD4 (#BE0003-3, Bio X Cell, Lebanon, NH, USA), 200 µg of anti-CD8 (#BE0061, Bio X Cell, Lebanon, NH, USA) and 200 µg of IgG (#BE0093, Bio X Cell, Lebanon, NH, USA) intraperitoneally 2 days prior to the injection of mEER cells, 2 days after and finally on the 7th day after the injection of mEER cells in mice. In addition, the mice were treated with or without 10μM of CBD every day and continued till Day 21. Tumors were measured with an external caliper, and the tumor volume was calculated as: (length x width²)/2. All experimental protocols were approved by the Institutional Animal Care and Use Committee of UCSD (#S16200) and maintained under specific pathogen-free conditions at Moores Cancer Center with the approval of the Institutional Animal Care and Use Committee of the University of California San Diego.

Flank tumors after treatment with CBD and vehicle from wildtype mice C57BL/6J mice were harvested on Day 15, minced, and resuspended by using Tumor Dissociation Kit (Miltenyi Biotec) diluted into DMEM for subsequent processing with the gentleMACS Octo Dissociator, according to the manufacturer’s recommendations for tumor dissociation into a single-cell suspension. Digested tissues were then passed through 70 µm strainers to produce a single-cell suspension. Samples were washed with PBS and processed for live/dead cell discrimination using Zombie viability stains (BioLegend). Cell suspensions were then washed with cell staining buffer/FACs Buffer (BioLegend) prior to cell surface staining, performed at the indicated antibody dilutions for 30 min at 4 °C, and protected from light. Cell surface staining was performed for 30 min at 4 °C with the following mouse antibodies from BioLegend: CD45 (#30-F11, 1:100), CD3 (#17A2, 1:100), CD8 (#53-6.7, 1:100), CD4 (#RM4-4, 1:100), CD19 (#6D5, 1:100), NK1.1 (#PK13, 1:100), Ly6C (#HK1.4, 1:100), CD11b (#M1/70, 1:100), CD11c (#N418, 1:100), MHCII (#M5/114.15.2, 1:100), Ly6G (#1A8, 1:100) and XCR1 (#ZET, 1:100). Stained cells were washed and then fixed with BD cytofix (#554655, BD Biosciences, New Jersey, USA) for 20 min at 4 °C, protected from light. Finally, cells were washed and resuspended in FACS buffer and acquired using the BD LSRII Fortessa. Downstream analysis was performed using FlowJo, (version 10.6.2). Absolute T-cell numbers per milligram of tumor were derived from CD45+ -cell counts in 100μL cell suspension divided by tumor weight.

Flank tumor tissues from wildtype mice C57BL/6J mice were collected and paraffin-fixed at the Day 22. Tissue blocks were prepared and processed for IHC analysis. The IHC analysis was performed for CD4+T cells, CD8+T cells and CD19+B cells as reported earlier (43). The Images were analyzed using the QuPath Software version 0.4.3.

The 5μm paraffin sections from flank tumor tissues from wildtype C57BL/6J mice were subjected for multiplex IHC analysis. First, the sections were baked at 60°C for 1 hour followed by tissue rehydration through successive steps of xylene, 100% ethanol, 95% ethanol, 70% ethanol and finally rinsing with distilled water. After rinsing the slides antigen retrieval was done by incubating them in a citrate based (pH 6.0) antigen unmasking solution (Vector Laboratories, Newark, CA, USA) at 95°C for 30 minutes. Slides were then cooled down to room temperature and were blocked using Peroxidase blocking, Bloxall Solution (Vector Laboratories, Newark, CA, USA) for 10 minutes. The slides were then washed with 1X TBST, followed by incubating with primary antibody anti-CD8 (Invitrogen, Waltham, MA, USA) for 1 hour, followed by washing with 1X TBST. Next, the slides were incubated with anti-Rat HRP Polymer secondary antibody (Cell IDX, San Diego, CA, USA) for 30 minutes followed by washing. Finally, the sections were incubated with fluorophore Opal 570 (Akoya Biosciences, Marlborough, MA, USA) for 10 minutes. The same incubation steps were repeated for the other primary antibodies such as anti-CD4 (Abcam, Cambridge, UK), anti-pp38 (Cell Signaling Technology, Danvers, MA, USA), anti-PanCK (Dako, Glostrup, Denmark) and anti-CD45 (Abcam, Cambridge, UK). The slides were washed with 1X TBST and incubated with secondary antibody anti-Rabbit HRP Polymer for 30 minutes. Slides were again washed with 1X TBST and incubated with fluorophore Opal 690, Opal 620, Opal 520 and Opal 780 (Akoya Biosciences, Marlborough, MA, USA) for 10 minutes respectively. After the final incubation with fluorophores, slides were washed with 1X TBST, followed by washing with distilled water and then incubation with DAPI (1µg/ml) for 15 minutes. Finally, slides were mounted on a coverslip with Vectashield Vibrance (Vector Laboratories, Newark, CA, USA). Images were analyzed for colocalization of CD4, CD8 and pp38 using the QuPath Software version 0.4.3 and protocol created by Lumanto et al. (44).

Statistical analysis was performed for three independent experiments by using GraphPad Prism 10.4.0 software.

Student’s t-test was performed to assess the statistical significance. P<0.05 or less was considered for statistical significance.

To determine the nature of CBD-mediated inhibition of tumor growth, cell proliferation was assessed in HPV-positive HNSCC by AquaBluer solution as well as a BrdU assay. It was observed that 10 μM of CBD treatment significantly decreased cell proliferation of HPV-positive HNSCC cells as compared to vehicle treated cells ( Figures 1A-D ). Next, we evaluated the ability of CBD to induce apoptosis in HPV-positive HNSCC cancer cells following treatment with 10 μM of CBD for 24 and 48 hours by Annexin-V/PI staining. A significant increase in the percentage of apoptotic cells upon treatment with 10 μM of CBD was observed as compared to the vehicle counterpart for UPCI: SCC154 cells ( Figure 1E ). In UD-SCC-2 cells, although we observed an increase in the percentage of apoptosis after treatment with 10 μM of CBD, but it was not statistically significant ( Figure 1F ). Enhanced cellular migration is a phenotypic characteristic of malignancy (45), thus, in this study, we interrogated the ability of CBD in inhibiting cellular migration by using the wound healing or scratch assay. The percentage of cellular migration of HPV-positive cell lines UD-SCC-2 and UPCI: SCC154 was determined after treatment with a vehicle and 10 μM of CBD for 12 and 24 hours. Wound healing assay showed CBD treatment significantly decreased the percentage of cellular migration as compared to the vehicle after 12 hours and 24 hours in HPV-positive HNSCC cells ( Figures 1G, H ). These results collectively demonstrate the anti-cancer properties of CBD in HPV-positive HNSCC cells by inhibiting proliferation and migration of HPV-positive HNSCC cells and by promoting apoptosis using in vitro assays.

It is well known that the mitogen-activating protein kinase (MAPK) pathway plays an essential role in cell proliferation, differentiation, and senescence (46). In a previous study by our group, we observed that stimulation of cannabinoid receptors by its agonists promotes the activation of p38 MAPK pathway (24). Previous reports suggest activation of MAPK pathway upon CBD stimulation promotes apoptosis (47, 48). In accordance with the previous studies, we also observed that CBD treatment (1-10 µM) increased the expression of MAPK markers such as pp38, pERK1/2, pJNK and pMK-2 in a dose-dependent manner after 15 minutes ( Supplementary Figures S1A, B ) and 30 minutes ( Figures 2A, B ) compared to vehicle treated HPV-positive HNSCC cells. These results suggested that stimulation with CBD led to the activation of MAPK pathway along with its other markers such as ERK1/2, JNK/SAPK and MK2 in HPV-positive HNSCC cells.

Next, to evaluate the anti-tumor activity of CBD in vivo, both immunocompetent and immunocompromised syngeneic mice models (wild-type C57BL/6J mice, athymic nude mice, and Rag1 KO mice) were used. In the immunocompetent syngeneic C57BL/6J mice model (wild-type), we observed that treatment with 10 μM of CBD significantly decreased the tumor volume as compared to the vehicle-treated group on Day 21 ( Figure 3A ). In contrast, no significant difference in tumor volume was observed between vehicle and 10 µM of CBD treated athymic nude mice and Rag1 KO mice on Day 21 ( Figures 3B, C ). These results suggested that the effect of CBD on tumors might be immune-modulated.

Next, to further evaluate the immune-modulatory activity of CBD, CD4+T and CD8+ T cell depletion was performed in immunocompetent C57BL/6J wild-type mEER mice model. As observed earlier, the mice treated with 10 µM of CBD showed a significant decrease in tumor volume at the end of Day 21 compared to vehicle-treated controls. In contrast, depletion of CD4+T cells resulted in an increase in tumor volume and CBD treatment enhanced the tumor growth in CD4-depleted mice suggesting tumor growth-promoting effects of CBD in the absence of CD4+T cells and a key role of CD4+T cells in CBD-mediated anti-tumor activity ( Figure 4A ). Next, we observed that depletion of CD8+T cells resulted in an increase in tumor volume, however, no significant difference in tumor volume was observed in CD8-depleted mice after CBD treatment ( Figure 4B ). However, mice treated with IgG antibody with or without treatment with 10 µM of CBD exhibit similar results as observed in mice treated with or without CBD ( Figure 4C ). Taken together, these results suggested that CBD-mediated tumor inhibition is mediated by both CD4+T and CD8+T cells, and that CBD can be growth stimulatory in vivo in the absence of CD4+T cells, suggesting a powerful influence of CD4+T cells in mediating CBD immune inhibition of tumor growth.

Next, we explored the role of CBD in modulating infiltration of immune cells in TIME, by immune-flow cytometry analysis on flank tumors of C57BL/6J wild-type mice treated with or without 10 μM of CBD ( Supplementary Figure S3A ). Tumors were harvested on Day 15 and single-cell suspensions were acquired from the harvested tumors and the cell surface was stained with the following mouse antibodies: immune cells (CD45+), T lymphocytes (CD3+, CD8+, and CD4+), B lymphocytes (CD19+), natural killer cells (CD3-NK1.1+), conventional Type 1 dendritic cells (cDC1) (CD11b-CD11c+MHCII+XCR1+), monocytic myeloid-derived suppressor cells (M-MDSCs) (CD11b+MHCII-Ly6CHighLy6G-) and polymorphonuclear MDSCs (PMN-MDSCs) (CD11b+MHCII-Ly6ClowLy6G+), macrophages (M1-like/anti-tumor: CD11b+Ly6C- MHCII+ and M2-like/pro-tumor: CD11b+Ly6C-MHCIIint). Flow cytometric analysis revealed a significant increase in the absolute count per milligram of the tumor tissue for CD3+ ( Figure 5A ), CD4+ ( Figure 5B ), and CD8+ ( Figure 5C ) T cells along with CD19+ B cells ( Figure 5D ), natural killer cells ( Figure 5E ), and M1-like macrophages ( Figure 5F ) in the CBD-treated mice as compared to the vehicle-treated mice. On the contrary, there was no significant difference for cDC1s, M-MDSCs, PMN-MDSCs, and M2-like macrophages in the CBD-treated mice group compared to the vehicle-treated mice ( Supplementary Figures S3B-E ).

Next, we validated the infiltration of CD4+ T cells, CD8+ T cells, and CD19+ B cells upon CBD stimulation by IHC analysis in tumor sections obtained from immune-competent C57BL/6J wild-type mEER mice. The tumors were harvested after Day 21 for flank tumors upon completion of the treatment (vehicle and 10 μM of CBD). IHC results indicated a significant increase in the percentage of CD4+T cells ( Figures 5G, H ), CD8+T cells ( Figures 5I, J ), and CD19+B cells ( Figures 5K, L ) in the tumor sections of 10 μM CBD-treated mice as compared to the vehicle counterparts.

Furthermore, we performed multiplex IHC to evaluate the infiltration of CD4+T cells and CD8+T cells along with activation of MAPK pathway in mice tumor sections obtained from vehicle and CBD-treated groups. We observed that increased accumulation of immune cell markers such as CD45, CD4+T cells, and CD8+T cells in CBD-treated mice tumor sections as compared to vehicle-treated counterparts. In accordance with our in vitro study, we also observed activation of MAPK pathway in CBD-treated mice tumor sections as evident from increased phosphorylation of p38 ( Figures 6A, B ). Along with these findings we also observed no significant difference between vehicle and CBD treated tumors for co-localization of panCK with pp38 ( Figure 6C ). On the contrary co-localization of CD4+T cells and CD8+T cells with pp38 were significantly higher in the CBD treated group compared to the vehicle treated group ( Figures 6D-F ). These results suggested the role of CBD in promoting colocalization of CD4+T cells and CD8+T cells with concurrent activation of pp38 MAPK which is related to cellular proliferation, apoptosis, differentiation, development, and inflammatory responses (47). Thus, CBD in the tumor microenvironment may promote direct activation of CD4+T cells and CD8+T cells and their interaction between immune cells and tumor cells.