But MOST interestingly ⭐, when we benchmarked iHOTT against patients treated with pembrolizumab as part of a clinical trial, we noted the *exact same* cell type shifts, validating that iHOTT recapitulates patient biology. This was an exciting finding ‼️

Our model preserved immune and tumor cell types after co-culture. We also saw immune activation and upregulation of GBM-linked cytokines like IL-6, IL-8, IL-10, and G-CSF, notably seen *only* when tumor cells and PBMCs were cultured together.

Most GBM models miss one of two things:
🛡️ a functional immune system
🧠 a human brain microenvironment

iHOTT has both.
🧫 We co-culture freshly isolated patient tumor cells + matched PBMCs on human cortical organoids. It’s patient-specific, matched, and immunocompetent.

Cre-dependent anterograde tracing demonstrates that thalamocortical axons make physical connections with cortical outer radial glia - a primate-enriched progenitor cell type central to cortical expansion. 🧠 But what types of cell-cell interactions mediate this contact?

Interestingly, severance ✂️ of the thalamic input midway through neurogenesis actually maintains these increased upper layer neurons. But how is early thalamic input changing the future fate of cortical cell types? Our work indicates communication with progenitors may be the answer…

Utilizing the human thalamocortical assembloid model, we show that thalamic input increases the generation of upper layer cortical neurons compared to cortex-only controls. 🧫 This work shows extrinsic cues like thalamic input can shape intrinsic cortical programs.

This led us to a model that indicates that glycolysis, through PPP, seems to restrict radial glia from generating oRGs, astrocytes, and inhibitory neurons too early in development. This may be why inhibition of glycolysis and PPP led to an increase of these cell types.

To test if PPP was guiding radial glia cell fate, we (painfully) extracted EGFP labeled radial from organoids and knocked down PPP genes. Indeed, same story. More oRGs, astrocytes, and inhibitory neurons paired with a decrease in PPP metabolite abundance.

This was fascinating because PPP also peaks in primary tissue at the same time as glycolysis. Was glycolysis working through the PPP? We tested this by inhibiting PPP with pharmacological inhibition, and also saw an increase in oRGs, astrocytes, and inhibitory neurons.

This was intriguing because glycolysis has been a flash point and somewhat controversial in organoids. Is it up in organoids? And if so, does it impact cell fate? We tested this by lowering glucose levels in organoid culture and measuring how cell types changed.