CN114657212A

CN114657212A - Macrophage for enhancing function based on gene editing metabolism, and preparation method and application thereof

Info

Publication number: CN114657212A
Application number: CN202210318487.2A
Authority: CN
Inventors: 张进; 王旭东
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-06-24

Abstract

The invention provides a macrophage for enhancing functions based on gene editing metabolism, a preparation method and application thereof, and relates to the technical field of biology. According to the method for improving the function of the macrophage by editing the metabolic pathway, the CRISPR screen method is used for screening metabolic genes influencing macrophage polarization, then the pluripotent stem cells or the monocytes with the metabolic genes knocked out are constructed, and the macrophages with different tumor killing capacities are obtained after induced differentiation. The invention provides application of a KEAP1 gene and/or an ACOD1 gene in regulation of macrophage M1 polarization and/or macrophage killing capacity. The tumor killing efficiency of the macrophage provided by the invention can be improved by 60%, the secretion of inflammatory factors and chemotactic factors higher than that of wild cells can be maintained in the co-culture process with tumor cells, and the macrophage can be used for preparing tumor treatment products.

Description

Macrophage for enhancing function based on gene editing metabolism, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a macrophage for enhancing functions based on gene editing metabolism, and a preparation method and application thereof.

Background

Macrophages are immune cells that have phagocytic and antigen-presenting functions. Engineered CAR-expressing macrophages have the ability to target killing of tumor cells expressing a certain antigen. CAR-M enters tumor tissue more readily than CAR-T cells, which is a natural advantage for solid tumors. In addition, the macrophage can also secrete inflammatory factors and chemotactic factors to regulate the tumor microenvironment and perform better anti-tumor effect with other immune cells in a synergistic way. Therefore, the method has good application prospect in the future. CAR-M cells differentiated from ipscs enable a monoclonal, highly homogeneous product and can ultimately yield sufficient numbers of cells for clinical therapy without limitation. Therefore, the iPSC differentiated CAR-M cell is one of the ideal choices for future immune cell products. However, clinical application of CAR-M cells faces a series of challenges, firstly its anti-tumor effect is not clearly superior to CAR-T, secondly its role in regulating the tumor microenvironment needs to be maintained for a longer time, and thirdly, there is currently no comprehensive study of genes affecting macrophage function.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

A first object of the present invention is to provide a method for improving macrophage function by editing metabolic pathway, so as to solve at least one of the above problems.

The second purpose of the invention is to provide the application of the gene in regulating and controlling the polarization and/or the killing capacity of macrophage M1.

The third objective of the invention is to provide a macrophage.

The fourth purpose of the invention is to provide a preparation method of the macrophage.

The fifth object of the present invention is to provide a pluripotent stem cell capable of differentiating the macrophage.

The sixth object of the present invention is to provide a monocyte capable of differentiating the macrophage.

The seventh purpose of the invention is to provide the application of the macrophage, the pluripotent stem cell or the monocyte in preparing products for treating tumors.

An eighth object of the invention is to provide a product for the treatment of tumors.

In a first aspect, the present invention provides a method for enhancing macrophage function by editing metabolic pathways, comprising the steps of:

metabolic genes influencing macrophage polarization are obtained through screening by a CRISPR screen method, then pluripotent stem cells or monocytes knocked out by the metabolic genes are constructed, and macrophages with improved or reduced tumor killing capacity are obtained after induced differentiation.

As a further aspect, the monocytes comprise THP-1 monocytes.

In a second aspect, the present invention provides the use of a gene comprising at least one of NDUFS, MPST, CYB561A, SLC4A, KEAP, NDST, PPA, leprl, NDUFB, OGFOD, ATP5, SLC16A, NFKB, PLPP, ASMTL, PIGF, ATP5, NR1H, acpp, ALDH9A, B3GNT, ASNSD, ST3GAL, NANS, EPN, CYP2R, ULK, PIP4K2, GCLM, CECR, TET, MFSD, ALG, start, ALDH, PPARD, GPD, AKR7A, IMPDH, gnp, PIK3, PIGT, SFXN, ALDH9A, SLC17A, SLC38A, SLCY, SLC x, folc, GCLC, ALDH, lac 18A, nacd, SLC 18, xca, xc, SLC39, xc, SLC39A, or kdod for modulating macrophage M polarization and/or macrophage killing ability.

As a further technical scheme, the KEAP1 gene is knocked out to inhibit the polarization of macrophages to the direction of M1, and/or the KEAP1 gene is knocked out to reduce the killing capacity of the macrophages;

the ACOD1 gene is knocked out to promote macrophage polarization to M1 direction, and/or the ACOD1 gene is knocked out to improve macrophage killing capacity.

In a third aspect, the invention provides a macrophage, wherein the ACOD1 gene of the macrophage is knocked out.

In a fourth aspect, the present invention provides a method for preparing macrophages, comprising the steps of:

obtaining pluripotent stem cells or monocytes with ACOD1 gene knockout, and obtaining macrophages after induced differentiation;

preferably, the monocytes comprise THP-1 monocytes.

In a fifth aspect, the present invention provides a pluripotent stem cell differentiated to give the above-mentioned macrophage, the pluripotent stem cell having a knockout of ACOD1 gene.

In a sixth aspect, the present invention provides a monocyte capable of differentiating to obtain the above-mentioned macrophage, wherein the monocyte has ACOD1 gene knockout.

In a seventh aspect, the invention provides the use of the above-mentioned macrophages, pluripotent stem cells or monocytes for the preparation of a product for the treatment of tumors.

In an eighth aspect, the invention provides a product for treating a tumor, the product comprising the above-described macrophage.

Compared with the prior art, the invention has the following beneficial effects:

according to the method for improving the function of the macrophage by editing the metabolic pathway, provided by the invention, metabolic genes influencing macrophage polarization can be found, and the macrophages with different tumor killing capacities can be obtained by knocking out the metabolic genes.

The invention provides application of a KEAP1 gene and/or an ACOD1 gene in regulation of macrophage M1 polarization and/or macrophage killing capacity. The research of the inventor finds that the KeAP1 gene is knocked out to inhibit the polarization of macrophages to the M1 direction and reduce the killing capacity of the macrophages; the ACOD1 gene has an inhibiting effect on the KEAP1 gene, and the ACOD1 gene is knocked out, so that the macrophage polarization towards the M1 direction can be promoted, and the macrophage killing capacity can be improved.

According to the macrophage provided by the invention, the ACOD1 gene of the macrophage is knocked out, the research of the inventor finds that the tumor killing efficiency of the macrophage can be improved by 60%, the macrophage can maintain the secretion of inflammatory factors and chemotactic factors higher than that of wild cells in the co-culture process with tumor cells, and the macrophage can be used for preparing tumor treatment products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Fig. 1 is a flow diagram of macrophage polarizing CRISPR screen;

FIG. 2 is a volcano plot of CD80-high and CD80-low sample enriched genes in CRISPR screen;

FIG. 3 shows the inhibition of M1 polarization and the expression of genes associated with inflammatory factors in the absence of KEAP1 in THP-1 derived macrophages;

FIG. 4 shows that M1 polarization was promoted in THP-1 derived macrophages in the absence of ACOD 1;

figure 5 is that M1 polarization was promoted in the absence of ACOD1 in iPSC-derived macrophages;

FIG. 6 shows that ACOD 1-depleted iMAC co-cultured with tumor cells can better maintain M1 polarization state;

FIG. 7 shows that ACOD 1-depleted iMAC is more phagocytic when co-cultured with tumor cells;

FIG. 8CD80-high and CD80-low cell populations are enriched for the list of genes corresponding to the top 20 gRNAs.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the specific conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the present application, the term "Macrophage" generally refers to a leukocyte derived from monocytes, located in peripheral blood, an inflammatory tissue. In animals, the phagocytic bacteria, dead cells and cell debris are mainly used for non-specific immune regulation (innate immunity), and then phagocytosed substances are digested and presented, and then lymphocytes and other immune cells are used for specific immune regulation (acquired immunity).

In the present application, the term "Chimeric Antigen Receptor (CAR)" generally means that it is composed mainly of three parts, an extracellular membrane-binding region, a transmembrane region and an intracellular signaling region. The extracellular domain is a single chain variable domain (scFv) with the function of targeting and binding to the TAA of tumor specific antigen. The transmembrane region is typically composed of an immunoglobulin superfamily, such as CD8 or CD 28. The intracellular signaling zone is mainly composed of an intracellular signaling domain of an activation receptor which can activate immune cells, such as a T cell-specific costimulatory factor (4-1BB or CD28) and a signaling activation zone CD3 zeta. After the immune cells loaded with the chimeric antigen receptor are specifically combined with the surface antigen of the tumor cells, the extramembranous antigen binding region transmits signals to the intracellular signal activation region, and then the immune cell activation reaction is started.

In the present application, the term "Induced Pluripotent Stem Cell (iPSC)" generally refers to a pluripotent stem cell having the potential to differentiate into various cells, which is obtained by transferring a pluripotent factor in an adult cell and then reprogramming an initial genome expression profile.

In the present application, the term "iMAC" generally refers to Macrophage derived from iPSC-induced differentiation.

The method can find out metabolic genes influencing macrophage polarization, and obtains macrophages with different tumor killing abilities by knocking out the metabolic genes.

In some preferred embodiments, the monocyte is a THP-1 monocyte.

The research of the inventor finds that the KeAP1 gene is knocked out to inhibit the polarization of macrophages to the M1 direction and reduce the killing capacity of the macrophages; the ACOD1 gene has an inhibiting effect on the KEAP1 gene, and the ACOD1 gene is knocked out, so that the macrophage polarization to the M1 direction can be promoted, and the macrophage killing capacity can be improved.

The research of the inventor finds that the tumor killing efficiency of the macrophage can be improved by 60%, the macrophage can maintain the secretion of inflammatory factors and chemotactic factors higher than that of wild cells in the process of co-culture with tumor cells, and the macrophage can be used for preparing tumor treatment products.

In addition, the prior art mainly improves the function of iMAC by modifying CAR, the invention can be combined with CAR, and the anti-tumor capability of iMAC can be further improved by knocking ACOD1 on the basis of CAR-iMAC.

obtaining pluripotent stem cells or mononuclear cells with ACOD1 gene knockout, and obtaining macrophages after induced differentiation;

preferably, the monocyte is a THP-1 monocyte.

The preparation method is simple, and can obtain macrophage with ACOD1 gene knockout.

In a fifth aspect, the present invention provides a pluripotent stem cell capable of differentiating into the above-mentioned macrophage, wherein the ACOD1 gene of the pluripotent stem cell is knocked out.

The macrophage provided by the invention has high tumor killing efficiency, and can be used for preparing products for treating tumors.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Example 1

Differentiation and polarization of THP-1 cells: on day 0, the well-titered Human CRISPR Metabolic Gene Knockout Library virus was infected at an MOI of 0.3 at 1.5X 10⁷A THP-1 cell. After 24 hours, fresh RPMI1640 medium containing 10% fetal bovine serum was changed and puromycin (final concentration 0.7. mu.g/ml) was added, the solution was changed every other day, and puromycin addition was continued for 7 days to completely kill the uninfected cellsViral THP-1 cells. The eighth day was changed to RPMI1640 medium containing 50ng/ml phorbol ester (PMA), and after 48h the supernatant was discarded, washed once with PBS and stimulated for 24h with medium containing 50ng/ml LPS and 50ng/ml IFN-r. Collecting cells, staining with anti-human CD80 flow antibody for 15min, separating two groups of CD80-high and CD80-low cells, extracting gene, constructing library, and sequencing, wherein the experimental process is shown in figure 1.

Analysis of the sequencing results (as shown at A, B in fig. 2 and 8) can see that the genes enriched in sample CD80-low (right) include ndefs 6, MPST, CYB561A3, SLC4a7, KEAP1, NDST1, PPA2, LEPREL2, ndefb 5, OGFOD2, ATP5H, SLC16a13, NFKB1, PLPP4, ASMTL, PIGF, ATP5B, NR1H3, acpp 2, ALDH9a1, and the like. Genes enriched in sample CD80-high (left side) include B3GNT8, ASNSD1, ST3GAL6, NANS, EPN2, CYP2R1, ULK1, PIP4K2A, GCLM, CECR1, TET3, MFSD3, ALG13, STARD13, ALDH2, PPARD, GPD2, AKR7a2, IMPDH2, GNPNAT1, and the like. Suggesting that the knockout of KEAP1 may inhibit macrophage polarization to M1.

After knocking out KEAP1 (designated as sgKEAP1-1 group, sgKEAP1-2 group and sgKEAP1-3 group, respectively, collectively referred to as sgKEAP1 group) from THP-1 cells, 3 gRNAs were designed, respectively, and 50ng/ml LPS and 50ng/ml IFN-r were added to stimulate, as shown in FIG. 3 (A in FIG. 3 is 0-24h flow-type results; B in FIG. 3 is 0-24h WT group and sgKEAP1 group CD80 expression amounts; C in FIG. 3 is 0-24h group, sgKEAP1-1 group, sgKEAP1-2 group and sgKEAP1-3 group inflammatory factors IL6, IL1B, CXCL9 and CXCL10 expression amounts), and WT (wild-type THP-1 cells, control group) were significantly decreased compared with KEAP1 cells (CD 80A in FIG. 3), and the results are consistent with those shown in FIG. 3B. The amount of inflammatory factor expression was also significantly reduced in the KEAP1 knockout macrophages compared to WT macrophages after stimulation with LPS and IFN-r, as shown by C in FIG. 3.

Secondly, it was found that ACOD1 promotes the production of Itaconate, which inhibits the activity of KEAP1 by alkylation. We also performed screen in iMAC cells similar to that in THP-1 cells. On day 0, the well-titered Human CRISPR Metabolic Gene Knockout Library virus was infected at an MOI of 0.3 for 1.5X 10⁷An iPS cell. After 24 hours, fresh mTeSR1 medium was changed and puromycin (final concentration 0.25. mu.g/ml) was added, and the solution was changed daily and puromycin was added continuously for 7 days to completely kill the uninfected iPS cells. iPS cells were digested on day 8 and started to differentiate into macrophages (imacs). On day 30, the cells were stimulated with medium containing 50ng/ml LPS and 50ng/ml IFN-r for 24 h. Collecting cells, staining with anti-human CD80 flow antibody for 15min, sorting two groups of CD80-high and CD80-low cells, extracting genes enriched in CD80-high including PIK3CA, PIGT, SFXN5, ALDH9A1, SLC17A9, SLC38A2, SLCY, GPX3, XRED2, GCLC, HADHA, ALDH18A1, NAA20, KDM6B, CBR4, DLST, SLC39A6, OXCT1, ACOD1 and MFSD9 (C in FIG. 8), and sequencing after gene library construction. Suggesting that the knockout of ACOD1 may promote macrophage polarization. We then started again with ACOD1 upstream of KEAP1 and studied its effect on M1 polarization. We constructed ACOD1 knock-out cells (marked as sgACOD1-2 and sgACOD1-3) in THP-1 and iPSC respectively, mRNA level and protein level prove that ACOD1 gene knock-out in THP-1 cells is successful (C and D in figure 4), and M1 polarization level and related functions are verified.

After adding 50ng/ml LPS and 50ng/ml IFN-r to THP-1 cells (WT group, sgACOD1-2 group and sgACOD1-3 group) for 24 hours, the stimulation was removed and the cells were replaced with fresh RPMI1640 medium, and the expression of CD80 was detected by flow-assay at day 0 (day 0), day 1 (day 1), day 2 (day 2), respectively, as shown in FIG. 4 (A in FIG. 4 is CD80 expression amount of macrophages in day 0 (day 0), day 1 (24 h), day 2 (48 h), Unstimulated group, WT group, sgACOD1-2 group and sgACOD1-3 group, wherein Unstimated group is wild-type THP-1 cells cultured in RPMI1640 medium all the time, and IFN-r group were not cultured, and IFN-r group was cultured under the same conditions as those in other three groups, and the statistical result is shown in FIG. 4C And sgACOD1-3 group was stimulated for 24 hours with 50ng/ml LPS and 50ng/ml IFN-r, respectively, the stimulation was removed, fresh RPMI1640 medium was replaced, cells were collected at day 0 (day 0), day 1 (day 1), and day 2 (day 2), respectively, and the amount of mRNA expressed by ACOD1 in each sample was measured; d in FIG. 4 shows that after macrophages of WT group, sgACOD1-2 group and sgACOD1-3 group are stimulated for 24h by adding 50ng/ml LPS and 50ng/ml IFN-r, cells are collected and the ACOD1 protein expression amount is detected by western blot; e in FIG. 4 shows the mRNA expression levels of inflammatory factors IL6, CXCL9, CXCL10 and CXCL11 before macrophage stimulation in WT group and sgACOD1-3 group, and after 2h, 8h and 24h of stimulation by adding 50ng/ml LPS and 50ng/ml IFN-r. ) It was found that CD80 expression of ACOD1 knock-out cells was consistently higher than that of WT cells (a in fig. 4 and B in fig. 4), and that inflammatory factors were also expressed higher than that of WT cells (E in fig. 4).

We obtained an ipod 1 gene 8bp base-deleted iPSC monoclonal cell (as shown in a in fig. 5), differentiated the iPSC into iMAC, and verified that the knock-out was successful at mRNA level (B in fig. 5) and intracellular average protein concentration level (C in fig. 5). 50ng/ml LPS and 50ng/ml IFN-r were added to iMAC for 24h of stimulation. The results are shown in FIG. 5 (A in FIG. 5 is a base sequence diagram and a primary sequencing peak diagram of the normal ACOD1 gene (WT ACOD1) and the ACOD1 gene (ACOD1-KO 3-5) deleting 8bp of bases after editing CRISPR Cas9 gene, and B in FIG. 5 is an iPSC cell (later labeled as ACOD1-KO 3-5) deleting 8bp of bases of WT iMAC and ACOD1 genes^-/-) iMAC, ACOD1 mRNA expression in cells at 6h and 24h before (nonstimulated) and after (3 h) LPS and 50ng/ml IFN- γ stimulation; c in FIG. 5 is WT iMAC and ACOD1 for mass spectrometric detection^-/-Average intracellular Itaconate concentration after 24h LPS and IFN- γ stimulation of iMAC; d in FIG. 5 is an unstained group (unstained), and WT iMAC and ACOD1^-/-iMAC is used for detecting the expression level of CD80 by flow under three conditions of unstimulation, 24h stimulation by 50ng/ml LPS alone and 24h stimulation by 50ng/ml LPS and 50ng/ml IFN-gamma; e in FIG. 5 is the statistics of the results of 3 experiments under the same conditions as D; f in FIG. 5 is WT iMAC and ACOD1^-/-iMAC detected mRNA expression of inflammatory factors IL6, IL1B, IL1A, IL23A, TNF α, chemokines CXCL9, CXCL10, CXCL11, chemokine receptor CCR7, M2 polarization-related inflammatory factors, chemokines IL6, TGF β, CCL2, CCL7 by qPCR 24 after LPS and IFN- γ stimulation for 24 h. ACOD1 in both LPS alone and IFN-r combination^-/-The expression level of CD80 was higher for iMAC than for WT iMAC (D and graph in FIG. 5)E in 5). After 24h stimulation with LPS and IFN-r, it was found to be ACOD1^-/-Most of the imacs expressed M1 polarization-related inflammatory factors higher than WT imacs (imacs differentiated from wild-type ipscs), whereas M2 polarization-related anti-inflammatory factors and receptor expression did not differ significantly (F in fig. 5).

Co-culture experiments of iMAC, Nalm6 and K562 tumor cells can show that ACOD1 knocked-out iMAC cells have stronger tumor killing capacity, as shown in figure 6 (A in figure 6 is WT iMAC and ACOD1)^-/-After iMAC is cultured with Nalm6 and K562 tumor cells for 24 hours at two effective target ratios of 3:1 and 5:1, the expression levels of M1 polarized markers CD80 and CD86 and M2 polarized markers CD163 and CD206 are detected in a flow mode; b in FIG. 6 is WT iMAC and ACOD1^-/-A statistical graph of the expression levels of M1 polarized markers CD80 and CD86 and M2 polarized markers CD163 and CD206 after iMAC is co-cultured with Nalm6 cells at a 5:1 effective target ratio for day 1, day 2 and day 3, respectively, for 24h and 72 h; c in FIG. 6 is WT iMAC and ACOD1^-/-After iMAC was co-cultured with Nalm6 cells for 24h at two effective target ratios of 3:1 and 5:1, mRNA expression of inflammatory factors IL6, IL1B, IL23A, TNF α, chemokines CXCL9, CXCL10, CXCL11, chemokine receptors CCR5, CCR7 in macrophages. ). After iMAC and Nalm6, K562 tumor cells were co-cultured for 24h, ACOD1^-/-The expression of both M1 polarized representative surface marker CD80 and CD86 was higher than WT iMAC for iMAC cells (ACOD1 knock-out iMAC cells), while both marker CD206 and CD163 were lower than WT iMAC for M2 polarized, with significant differences (a in fig. 6 and B in fig. 6). Illustrating that ACOD1 is present during co-culture with tumor cells^-/-iMAC can be better maintained in M1 polarization state, has stronger tumor killing ability. ACOD1 after 24h of cocultivation with Nalm6 cells^-/-inflammatory factors, chemokines and chemokine receptors were also expressed higher than WT imacs for imacs (see C in fig. 6).

FIG. 7 shows the results of experiments with K562 and AspC-1 cells (A in FIG. 7 is WT iMAC and ACOD1^-/-The iMAC, K562 and AspC-1 cells are co-cultured for 24h, and the result of phagocytosis, namely double positive cells, is detected in a flow mode; b in FIG. 7 is the statistics of the results of 3 experiments under the same experimental conditions as A; c in FIG. 7 is WT iMAC and ACOD1^-/-iMAC andafter K562 and Nalm6 cells are co-cultured for 24 hours, the amount of the surviving tumor cells is detected by a luciferase experiment, so that the killing rate of the tumor cells is obtained. ) ACOD1 compared to WT iMAC^-/-The proportion of double positives in iMAC is higher, the difference being significant, that is to say ACOD1^-/-imacs are more potent in phagocytosing tumor cells (e.g., a in fig. 7 and B in fig. 7). The experiment of tumor killing co-cultured with K562 and Nalm6 cells also shows that ACOD1^-/-iMAC has a stronger tumor killing ability (C in fig. 7).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for enhancing macrophage function by editing metabolic pathways, comprising the steps of:

2. The method of claim 1, wherein the monocytes comprise THP-1 monocytes.

3. Use of a gene for modulating macrophage M polarization and/or macrophage killing ability, wherein the gene comprises at least one of NDUFS, MPST, CYB561A, SLC4A, KEAP, NDST, PPA, leprl, ndifb, OGFOD, ATP5, SLC16A, NFKB, PLPP, ASMTL, PIGF, ATP5, NR1H, acpp, ALDH9A, B3GNT, ASNSD, ST3GAL, NANS, EPN, CYP2R, ULK, PIP4K2, GCLM, CECR, TET, MFSD, ALG, STARD, ALDH, PPARD, GPD, AKR7A, IMPDH, gnnat, PIK3, PIGT, SFXN, ALDH9A, SLC17A, SLC38A, SLCY, GPX, foxr, GCLC, dha, ALDH18A, SLC 6, xca, xc, dcb 39A, afl, or afl.

4. The use according to claim 3, wherein the KEAP1 gene knockout inhibits macrophage polarization to M1 and/or the KEAP1 gene knockout reduces macrophage killing;

5. A macrophage, wherein the ACOD1 gene of said macrophage is knocked out.

6. The method of producing macrophages according to claim 5, comprising the steps of:

preferably, the monocytes comprise THP-1 monocytes.

7. A pluripotent stem cell capable of differentiating into a macrophage according to claim 5, wherein the ACOD1 gene of the pluripotent stem cell is knocked out.

8. A monocyte cell capable of differentiating to give a macrophage according to claim 5, wherein the monocyte cell has an ACOD1 gene knockout.

9. Use of the macrophage of claim 5, the pluripotent stem cell of claim 7 or the monocyte of claim 8 in the manufacture of a product for treating a tumor.

10. A product for use in the treatment of tumors, said product comprising the macrophage of claim 5.