Aim 3: Impact of tumor growth on lymphoid tissue organization
and function
Early studies have described structural aberrations in murine tumor
draining lymph nodes (16) and
several studies now describe systemic dysfunction in patient PBMC.
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Organization and immune activity of PBMC from cancer
patients in hLToC: Potential cancer types being considered are
melanoma (high mutation load and immunogenicity, longest clinical
experience with checkpoint blockade, potential for successful
collaboration) and lymphoma (disruption in lymph node structure are
key feature for differential diagnosis). However, any solid tumor
could potentially be used, particularly if samples are limiting. With
the exception of lymphoma, we will not test other hematological
malignancies at this stage as the presence of tumor cells within the
PBMC would confound the interpretation of results. Patient PBMC will
be obtained via pre-existing clinical collaborations (David Chou,
clinical fellow in Ingber lab) and new ones are being initiated with
my mentor’s colleagues and my previous colleagues at Dana Farber
Cancer Center. We will test 3D organization by imaging and the
expression of previously discussed (g) T cell coinhibitory receptors
(imaging if reagents and biology permit, else flow cytometry) and
stimulation with OKT3 vs OKT3+checkpoint blockade. These experiments
could potentially reveal biomarkers for diagnosis as well as response
to therapy not evident in patient PBMC.
Tumor interaction with organized immune structures can occur in
different ways. Tumors extravasate into lymphatics and establish
micrometastases in the lymph node. Tertiary lymphoid tissue can also be
seen within solid tumors and plays an active role in tumor growth,
immunosuppression and therapeutic responses. Thus, here we explore two
different models of tumor-lymphoid tissue interaction
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Impact of lymph drainage from a tumor on the
organization and activity of the lymph node: a microfluidic model
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Encapsulation of lymphoid tissue module in a solid tumor
to model tumor associated tertiary lymphoid structures. Depending on
feasibility, large number of cells from tumor cell lines or patient
specific tumors will be mixed with the lymphocytes to create a 3D
representation of tumor stroma with lymphocytes. Lymphocyte
organization, polarization and activation will be compared to
“healthy” If we are able to obtain matched patient tumor samples, we
will grow a solid tumor from the melanoma cells in combination with
the hLToC. Several possible combinations are possible but first we
will attempt the growth of unfractionated tumor sample (melanoma,
immune infiltrate and endothelial cells) in a neighboring channel (can
be seeded with <10ul volume, therefore even biopsies can be
used) separated from the lymphoid tissue by a lymphatic monolayer.
Impact on lymphoid tissue organization, and trafficking of labeled
cells from the hLN-on-chip to the cancer can be studied. Further
impact on inhibitory receptor and the effect of blocking function on
immune stimulation will also be studied as previously discussed. These
data will provide a basis for further iteration (tumor fractionation,
labeling, chip design) to study melanoma micrometastases in the LN,
the difference in response of tumor infiltrating lymphocytes and lymph
node lymphocytes to therapy and the construction of a lymph node
draining a microfluidcally connected tumor via a lymphatic vessel. In
case matched patient samples cannot be obtained other strategies such
as HLA typed human cell lines or using hematological malignancies to
obtain cancer cells along with PBMC could be considered.