Journal: Journal for Immunotherapy of Cancer

Article Title: Elicitation of stem-like CD8 + T cell responses via lymph node-targeted chemoimmunotherapy evokes systemic tumor control

doi: 10.1136/jitc-2022-005079

Figure Lengend Snippet: The 30 nm poly(propylene sulfide) NPs increase accumulation of encapsulated small molecule in late but not early stage E0771 TNBC tumors, facilitate sustained release of PXL with sustained cytotoxic and immune checkpoint activation effects, and improve tumor control from systemic chemotherapy. (A) E0771 tumor growth following implantation in the fourth mammary fat pad. (B) Ex vivo image of E0771 tumor microenvironment and proximal (tumor-draining) inguinal LN in the fourth MFP on day 11 post-implant. (C) Schematic of PPS-NP structure and loading of small molecules into hydrophobic NP core. (D) Fluorescent Cy5.5 model cargo-loaded NP (Cy5.5-NP) were administered intravenously into mice bearing d10 or d25 E0771 tumors and mice were sacrificed 24 hours later for tissue imaging. (E) IVIS images and (F) quantification of Cy5.5 content in tumors 24 hours post-intravenous injection of Cy5.5-NP. (G) Hydrodynamic diameter of unloaded (24.9±6.6 nm) or PXL-loaded (26.9±6.9 nm) NP analyzed via DLS. (H) Per cent of initial loaded PXL (starting concentration 1 mg/mL) amount remaining in NP or free during in vitro release with dialysis at 37°C. (I) Viability of E0771 tumor cells in vitro following 48 hours treatment with various concentrations of PXL in NP or free as determined via alamarBlue assay. (J) PD-L1 expression on E0771 cells treated with indicated concentrations of free PXL for 24 hours in vitro as determined using flow cytometry. (K) E0771 tumor-bearing mice (5×10 5 E0771 cells implanted s.c. in the fourth MFP) were treated intravenously on days 7, 10, 14, and 17 post-tumor implantation with PXL (7 mg/kg (140 μg) total dose) formulated in 35 mg/mL PPS-NP (PXL-NP) or Cremophor/Ethanol (free PXL), or corresponding saline, Cremophor/ethanol, and unloaded NP controls. (L) E0771 tumor growth in response to treatment. (M) Mice from (L) were euthanized on day 25 post-implant and tumors processed and analyzed using flow cytometry for PD-L1 expression on CD45– tumor cells. n=3–4 mice per treatment group. Statistical analyses by unpaired two-tailed t-test or one-way or two-way analysis of variance with Tukey’s test. Data represented as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001. Cy5.5, Cyanine5.5-carboxylic acid; DLS, dynamic light scattering; i.v., intravenous; IVIS, in vivo imaging system; MFP, mammary fat pad; PD-L1, programmed death ligand 1; PPS-NPs, poly(propylene sulfide) nanoparticles; PXL, paclitaxel; s.c., subcutaneous; TdLN, tumor-draining lymph node; TNBC, triple negative breast cancer.

Article Snippet: Mice were euthanized 24 hours post-injection, and tissues imaged using the IVIS Spectrum CT (PerkinElmer).

Techniques: Activation Assay, Control, Ex Vivo, Imaging, Injection, Concentration Assay, In Vitro, Alamar Blue Assay, Expressing, Flow Cytometry, Tumor Implantation, Saline, Two Tailed Test, In Vivo Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Elicitation of stem-like CD8 + T cell responses via lymph node-targeted chemoimmunotherapy evokes systemic tumor control

doi: 10.1136/jitc-2022-005079

Figure Lengend Snippet: LN accumulation is enhanced by NP encapsulation for model small molecular cargo Cy5.5 and locoregional compared with systemic administration for both Cy5.5 and aPD-1-AF610 mAb. (A) Schematic of biodistribution experiment in which Cy5.5 encapsulated in 35 mg/mL PPS-NP (AF555-labeled) or free (in 20% DMSO/saline), or aPD-1 mAb labeled with AF610, was injected intravenously (NP)/i.p. (mAb), or i.d. in the ipsilateral flank co-draining to d11 E0771 TdLNs, and 24 hours later TdLNs analyzed for NP (AF555), Cy5.5, or aPD-1 (AF610) fluorescent signal. (B) Representative IVIS images and (C) quantification of NP (AF555) fluorescent signal in tumor-draining and non-draining LNs from intravenous or i.d. administration. (D) Representative IVIS images and quantification of Cy5.5 fluorescent signal in tumor-draining and non-draining LNs of free or NP-formulated Cy5.5 24 hours after (E) intravenous and (F) i.d. administration. (G) Representative IVIS images and (H) quantification of aPD-1 (AF610) levels in tumor-draining and non-draining LNs of d11 E0771-bearing mice 24 hours after i.p. or i.d. administration. n=4–5 mice per treatment group. Statistical analyses by two-way analysis of variance with Sidak’s test. Data represented as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001. AF, Alexa Fluor; aPD-1, anti-programmed death-1; Cy5.5, Cyanine5.5-carboxylic acid; DMSO, dimethyl sulfoxide; i.d., intradermal; i.p., intraperitoneal; i.v., intravenous; IVIS, in vivo imaging system; mAb, monoclonal antibodies; nTdLN, non-tumor draining lymph node; PPS-NP, poly(propylene sulfide) nanoparticles; TdLN, tumor-draining lymph nodes.

Article Snippet: Mice were euthanized 24 hours post-injection, and tissues imaged using the IVIS Spectrum CT (PerkinElmer).

Techniques: Encapsulation, Labeling, Saline, Injection, In Vivo Imaging, Bioprocessing

Journal: Journal for Immunotherapy of Cancer

Article Title: Elicitation of stem-like CD8 + T cell responses via lymph node-targeted chemoimmunotherapy evokes systemic tumor control

doi: 10.1136/jitc-2022-005079

Figure Lengend Snippet: Dose reduction study reveals benefit of PXL-NP/aPD-1 combination chemoimmunotherapy directed to TdLNs by locoregional administration i.d. compared with i.t. on improving systemic tumor control and animal survival associated with increased mobilization of Tcf1+ stem like cells into the circulation. (A) On day 11 post E0771 tumor implant, mice were injected with 30 nm TRITC-dextran (0.05 mg in 10 µL saline) i.t. and AF647-labeled PPS-NPs (20 µL, 40 mg/mL) either i.t. or in the i.l. flank and sacrificed 4 hours post-injection for tissue imaging via IVIS CT. (B) Schedule for the therapeutic study, primary E0771 tumors were implanted as previously on day 0 and secondary tumors implanted in the contralateral fat pad on day 7. Mice were treated with PXL-NP i.l. or i.t. on days 7, 10, 14, and 17 post-tumor implantation (10, 2, 0.2 mg/kg total PXL dose) formulated in 35 mg/mL PPS-NP (PXL-NP) in combination with i.t. administration on days 10, 14, and 17 of aPD-1 (5 mg/kg mAb dose) formulated in saline. Blood was drawn on day 18 post-primary tumor implant and analyzed via immune phenotyping. (C) Ki67+ and (D) PD-1+ frequency of circulating CD8+T cells. (E) Tcf1+Tim3– stem-like and Tcf1–Tim3+ terminally differentiated cell frequencies of circulating PD-1+ CD8+ T cells. (F–G) E0771 primary or secondary tumor growth in response to saline or combination of unloaded NP and isotype mAb until first animal reaching humane endpoint. (H) Survival of mice treated in (F–G). (I–J) Primary and secondary tumor volumes of PXL-NP i.t. treated groups. (K–L) Primary and secondary tumor volumes of PXL-NP i.l. treated groups. (M–N) Comparison of primary and secondary tumor volumes of mice treated in (B–L) i.l. or i.t. with 10 mg/kg (P–Q) 2 mg/kg or (S–T) 0.2 mg/kg PXL-NP in combination with aPD-1 i.t. (O, R, U) Survival of mice treated in (B–L) i.l. or i.t. with (O) 10 mg/kg (R) 2 mg/kg or (U) 0.2 mg/kg PXL-NP in combination with aPD-1 i.t. n=5–6 mice per treatment group. Statistical analysis by one-way or two-way analysis of variance with Tukey’s test and log-rank (Mantel-Cox) test for survival analysis. Data represented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. aPD-1, anti-PD-1; i.l., ipsilateral; i.t., intratumoral; IVIS, in vivo imaging system; mAb, monoclonal antibodies; PD-1, programmed death-1; PPS-NP, poly(propylene sulfide) nanoparticles; PXL, paclitaxel; TdLN, tumor-draining lymph nodes.

Article Snippet: Mice were euthanized 24 hours post-injection, and tissues imaged using the IVIS Spectrum CT (PerkinElmer).

Techniques: Control, Injection, Saline, Labeling, Imaging, Tumor Implantation, Comparison, In Vivo Imaging, Bioprocessing