Implications on immune therapy

Our findings have broad implications on cancer immunotherapy. We showed that early tumor biopsies may not clearly indicate patient response rate. T cells in the tumor microenvironment showed no significant difference in the particular activation phenotypes tested. Nor were the phenotypes of myeloid cells significantly different at early stages. This finding reveals the importance of looking at the bigger picture of immune cell dynamics in the tumor microenvironment in order to ascertain whether a patient will respond to therapy. Potentially, tools need to be developed which would include immune cells dynamics as a prognostic indicator of response rates.

6. Appendix

Supplementary Movie 1. Dynamic immune cell motility patterns in tumor bearing mice. Activated T cells (green) as well as non-activated T cells (red) engaged in a heterogeneous mixture behavior patterns with both dendritic cells (white) and tumor nodules (blue) in tumor bearing mice. Some T cells engage the tumor. Other T cells engaged dendritic cells, while others move quickly in and out of frame without engaging any targets. This movie is one representative 45-minute-long movie from a total of four movies taken from four different mice ranging from 45 minutes to one hour long. This movie was taken using 20X objective (0.70 NA), Z-step size = 2 μm, utilizing sequential scanning with a 20 second scan time.

Supplementary Movie 2. Dynamic immune cell behaviors in tumor bearing mice receiving anti-PD-L1 antibody blockade. After 8 days of anti-PD-L1 antibody therapy, activated T cells (green) and non-activated T cells (red) exhibit complex behavior patterns with dendritic cells (white) and tumor nodules (blue). T cells demonstrate a mixture of behaviors ranging from contacting the tumor nodule, dendritic cells, other T cells, or no other visible cell type. Movie is one representative one-hour-long movie from a total of four movies taken from four different mice, taken using a 20X objective (0.70 NA), Z-step size = 2 μm, utilizing sequential scanning with a 20-second scan time.

Supplementary Movie 3. Interactions in the tumor microenvironment. Complex interactions in the tumor microenvironment were broken down using a novel imaging-based analysis technique (Figure 7B,C). Cells with persistent motility

characterized by overlapping binarized fluorescence were enumerated and interactions, defined as persistent motility within one T cell length of a second cell type, were quantified. Cells were categorized as interacting with dendritic cells alone (middle bottom), tumor alone, dendritic cell and tumor together (left middle), or neither dendritic cell nor tumor (upper right corner). Cells without overlap (upper left) were motile and did not stably interact with a partner. This movie is one representative one-hour-long movie from a total of four movies taken from four different mice. The movie was taken using a 20X objective (0.70 NA), Z-step size = 2 μm, utilizing sequential scanning with a 20 second scan time.

Supplementary Movie 4. Activated T cells engaged in unstable interactions with dendritic cells in the tumor microenvironment. Activated T cells (green) from control mice made unstable, sliding interactions with dendritic cells (red) surrounding the tumor (blue). One representative 45-minute-long movie taken of four one hour long movies from four different mice is shown. Movies were taken using an 20X objective (0.70 NA) , Z-step size = 2 μm, utilizing sequential scanning with a 20 second scan time.

Supplementary Movie 5. Activated T cells engaged in stable interactions with dendritic cells in the tumor microenvironment during PD-L1 blockade.

Activated T cells (green) from mice receiving one week of anti-PD-L1 antibody therapy made strong, stable engagement with dendritic cells (red) in surrounding the tumor (blue). One representative one-hour-long movie taken of four from four different mice. Movies were taken using an HCPL APO 20X/0.70 NA objective, Z-step size = 2 μm, sequential scanning using a 20-second scan time.

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8. Vita

Todd Jacob Bartkowiak was born on September 19, 1986 in Houston, Texas to James and Renalda Bartkowiak. After completing high school at Klein High School in Klein, Texas in 2005, he entered Sam Houston State University in Hunstville Texas. In May of 2008, he received his Bachelor of Science degree with a major in Biology and a minor in Chemistry. For two and a half years, Todd worked as a research assistant in the Diagnostic Sciences department at the University of Texas Health Science Center, Houston Texas. In August of 2011, Todd entered the University of Texas Health Science Center at Houston Graduate School of Biomedical Sciences. Todd completed this thesis under the mentorship of Dr.

Tomasz Zal in the Immunology Department at the University of Texas, MD Anderson Cancer Center.

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