# Programmed Cell Removal (PrCR) by Macrophages: recognition and phagocytosis of target cells

> **NIH NIH R01** · STANFORD UNIVERSITY · 2022 · $404,694

## Abstract

Project Summary
Macrophage-mediated programmed cell removal (PrCR) allows clearance of living cells. We have shown that
this phagocytic process can eliminate cancer cells that present an ‘eat me’ signal and have their dominant 'don't
eat me' molecules blocked. We then further extended this to the clearance of other pathogenic or ‘expired’ cells.
The key novel findings of our recent studies are: 1. Activated macrophages produce and secrete calreticulin
(CRT); 2. Secreted CRT binds to surface asialoglycans on target cells to create ‘eat me signals' for
macrophages. 3. Cancer cells and neutrophiles modulate their surface to expose asialoglycan binding sites for
CRT that acts as an ‘eat me’ signal for macrophages. We propose that by binding both to asialoglycans and to
pro-phagocytic receptors on macrophages (CD91/LPR1), CRT can bridge target cells to macrophages for
clearance via PrCR. CRT is normally a resident ER protein containing a C-terminal KDEL retention signal, but
it’s been shown that in dying cells CRT can be translocated to the cell surface. We found that upon macrophage
activation via toll-like receptors (TLR), CRT can both translocate to the cell surface and be secreted, leading to
increased PrCR of either WT (dying) or Bcl-2+ (viable) peritoneal neutrophils and cancer cells on which the ‘don’t
eat me’ signal CD47 is either absent or blocked. Based on these findings we proporse: (1) to elucidate the
signals affecting the macrophage that result in CRT translocation to the cell surface and secretion of soluble
forms of CRT; (2) to elucidate the mechanisms regulating the availability of asialoglycan-containing binding sites
for CRT on target cells. Elucidating the individual mechanisms in macrophages and target cells required for PrCR
and understanding the cross-talk within macrophage:target-cell interaction can have broad therapeutic
implications. In Aim 1 we will investigate which signals stimulate macrophages to increase cell surface
expression and secretion of CRT for PrCR and the heterogeneity of macrophages that can carry out PrCR in
vitro and sterile inflammation in vivo. In Aim 2 we will employ proteomic analysis to define and characterize the
different proteoforms of CRT originating in macrophages before or after stimulation: the ER form vs. cell-surface-
bound, vs. soluble secreted CRT, vs. CRT that is bound to asialoglycans on target cells. We have preliminary
evidence that the secreted form of CRT does not contain the KDEL motif and that potential proteolytic processing
leads to the formation of the different CRT forms. Lastly in Aim 3 we will study the initiating signaling events and
enzymes that affect the addition or removal of sialic acid, the activity of which determines the level of exposed
asialoglycans and thus the binding of CRT to the surface of cells destined for elimination. These studies will shed
light on a novel mechanism by which macrophages detect cells that are to be removed from the body. Our
findings will ha...

## Key facts

- **NIH application ID:** 10328484
- **Project number:** 5R01AI143889-03
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** IRVING L. WEISSMAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $404,694
- **Award type:** 5
- **Project period:** 2020-02-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10328484

## Citation

> US National Institutes of Health, RePORTER application 10328484, Programmed Cell Removal (PrCR) by Macrophages: recognition and phagocytosis of target cells (5R01AI143889-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10328484. Licensed CC0.

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