Figure 2.

Local increases in CTL markers in tumor lesions after systemic application of the CKM. (A) CD8⍺ and granzyme B (GZMB) transcripts (normalized for HPRT1 and shown as mean and median (MED) fold-increases over pre-CKM baseline for each patient; N=6); were measured using RT-qPCR (see online supplemental table 1 for raw data). Statistically significant increases in CD8α mRNA and GZMB mRNA following CKM treatment were observed. In contrast, FOXP3 message trended down following CKM. Statistically significant increases in ratios of CD8⍺/FOXP3 and GZMB/FOXP3 were also observed. Statistical significance by a two-sided one-sample t-test of the log-fold change was determined to be p≤0.084. (B) (top) Representative biopsy image stained with a multispectral panel consisting of GZMB, CD8, CD3, PD-1, and FOXP3 and tumor compartment marker, AE1AE3. The multiplex image depicts the spatial layout of the tumor environment, with monoplex images shown below. CTLs were defined as CD3⁺CD8⁺, Tregs were defined as CD3⁺FOXP3⁺. Examples of colocalization of biomarkers are depicted on the right. (bottom) Image analysis of biopsy pairs demonstrating a statistically significant difference is shown as mean (SD). The table describes the changes in the ratios of CTL/Treg and PD-1⁺CTL/Treg significantly increased in the post-treatment versus the pretreatment tumor tissues. P value≤0.084, determined by a two-sided one-sample t-test of the log-fold change was considered as statistically significant. CKM, chemokine modulatory; CTL, cytotoxic T lymphocytes; mRNA, messenger RNA; PD-1, programmed death-1; RT-qPCR, real-time quantitative polymerase chain reaction; Treg, regulatory T cells.